U.S. patent application number 16/226344 was filed with the patent office on 2019-08-01 for methods of microbial treatment of poultry.
This patent application is currently assigned to Church & Dwight Co., Inc.. The applicant listed for this patent is Church & Dwight Co., Inc.. Invention is credited to Evan Hutchison, Joshua Rehberger, Thomas Rehberger, Alexandra Smith.
Application Number | 20190231828 16/226344 |
Document ID | / |
Family ID | 65242322 |
Filed Date | 2019-08-01 |
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United States Patent
Application |
20190231828 |
Kind Code |
A1 |
Rehberger; Thomas ; et
al. |
August 1, 2019 |
METHODS OF MICROBIAL TREATMENT OF POULTRY
Abstract
Disclosed are methods of administering one or more Bacillus
strains to poultry. The Bacillus strains improve bacterial
homeostasis in the gastrointestinal tract by inhibiting bacterial
pathogens such as E. coli and Clostridium. Administering the
Bacillus strains also improves performance such as weight gain and
feed conversion. Useful combinations of Bacillus strains and
methods of using one or more Bacillus strains are also
provided.
Inventors: |
Rehberger; Thomas;
(Wauwatosa, WI) ; Hutchison; Evan; (Milwaukee,
WI) ; Smith; Alexandra; (Greendale, WI) ;
Rehberger; Joshua; (Milwaukee, WI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Church & Dwight Co., Inc. |
Princeton |
NJ |
US |
|
|
Assignee: |
Church & Dwight Co.,
Inc.
Princeton
NJ
|
Family ID: |
65242322 |
Appl. No.: |
16/226344 |
Filed: |
December 19, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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15268104 |
Sep 16, 2016 |
10201574 |
|
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16226344 |
|
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62219433 |
Sep 16, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A23K 10/18 20160501;
A23K 10/10 20160501; A61K 35/742 20130101; A61K 38/164 20130101;
A23K 50/70 20160501; A61K 2035/11 20130101; A23K 50/75
20160501 |
International
Class: |
A61K 35/742 20060101
A61K035/742; A23K 10/18 20060101 A23K010/18; A23K 10/10 20060101
A23K010/10; A61K 38/16 20060101 A61K038/16; A23K 50/70 20060101
A23K050/70; A23K 50/75 20060101 A23K050/75 |
Claims
1. A microbial composition comprising at least one isolated
Bacillus strain chosen from at least one of strains Bacillus
subtilis 1104, deposited as NRRL B-67258; Bacillus subtilis 1781,
deposited as NRRL B-67259; Bacillus subtilis 747, deposited as NRRL
B-67257; Bacillus subtilis 1541, deposited as NRRL B-67260; and
Bacillus subtilis 2018, deposited as NRRL B-67261; present in a
concentration of about 10.sup.5 CFU/gram to about 10.sup.12
CHU/gram, wherein the at least one isolated Bacillus strain
inhibits a pathogen chosen from at least one of E. Coli and
Clostridium in an animal
2. The microbial composition of claim 1, wherein the composition is
a freeze-dried composition.
3. The microbial composition of claim 2, further comprising a
carrier.
4. The microbial composition of claim 3, wherein the carrier is
selected from a group consisting of: whey, maltodextrin, sucrose,
dextrose, limestone, rice hulls, and sodium silica aluminate.
5. The microbial composition of claim 4, wherein the carrier is in
the physical form is selected from a group consisting of: a
powdered solid, a liquid, and a gel.
6. An animal feed comprising the microbial composition of claim 2,
wherein the microbial composition has concentration of the at least
one isolated Bacillus strain in the composition of about
1.times.10.sup.8 CFU/g.
7. The composition of claim 4, wherein the composition further
comprises the carrier in the physical form selected from a group
consisting of: a liquid, and a gel; and wherein the composition has
concentration of the at least one isolated Bacillus strain in the
composition of about 1.times.10.sup.8 CFU/g.
8. A direct fed microbial composition comprising at least one
isolated Bacillus strain is chosen from at least one of strains
Bacillus subtilis 1104, deposited as NRRL B-67258; Bacillus
subtilis 1781, deposited as NRRL B-67259; Bacillus subtilis 747,
deposited as NRRL B-67257; Bacillus subtilis 1541, deposited as
NRRL B-67260; and Bacillus subtilis 2018 deposited as 8-67261
present in a concentration of about 10.sup.5 CFU/grain to about
10.sup.12 CFU/gram wherein the at least one isolated Bacillus
strain inhibits a pathogen chosen from at least one of E. Coli and
Clostridium in an animal.
9. The direct fed microbial composition of claim 8, wherein the
composition comprises a plurality of isolated Bacillus strains
chosen from the strains Bacillus subtilis 1104, deposited as NRRL
13-67258; Bacillus subtilis 1781, deposited as NRRL 13-67259;
Bacillus subtilis 747, deposited as NRRL B-67257; Bacillus subtilis
1541, deposited as NRRL R-67260; and Bacillus subtilis 2018,
deposited as NRRL B-67261.
10. The composition of claim 8, wherein the at least one isolated
Bacillus strain is chosen from at least one of strains Bacillus 747
(NRRL B-67257) or a strain having all of the identifying
characteristics of Bacillus 747 (NRRL B-67257), Bacillus strain
1104 (NRRL B-67258) or a strain having all of the identifying
characteristics of Bacillus strain 1104 (NRRL B-67258), Bacillus
strain 1781 (NRRL B-67259) or a strain having all of the
identifying characteristics of Bacillus strain 1781 (NRRL B-67250),
Bacillus strain 1541 (NRRL B-67260) or a strain having all of the
identifying characteristics of Bacillus strain 1541 (NRRL B-67260),
and Bacillus strain 2018 (NRRL B-67261) or a strain having all of
the identifying characteristics of Bacillus strain 2018 (NRRL
B-67261).
11. The composition of claim 10, further comprising a carrier.
12. The composition of claim 11, wherein the carrier is selected
from a group consisting of: whey, maltodextrin, sucrose, dextrose,
limestone, rice hulls, and sodium silica aluminate.
13. A direct fed microbial composition comprising at least one
isolated Bacillus strain is chosen from at least one of strains
Bacillus subtilis 1104, deposited as NRRL B-67258, Bacillus
subtilis 1781, deposited as NRRL B-67259; Bacillus subtilis 747,
deposited as NRRL B-67257, Bacillus subtilis 1541, deposited as
NRRL B-67260; Bacillus subtilis 1999, deposited as NRRL B-67318;
and Bacillus subtilis 2018, deposited as NRRL B-67261 present in a
concentration of about 10.sup.5 CFU/gram to about 10.sup.12
CFU/gram wherein the at least one isolated Bacillus strain inhibits
a pathogen chosen from at least one of E. Coli and Clostridium in
an animal.
14. The direct fed microbial composition of claim 13, wherein the
composition comprises a plurality of isolated Bacillus strains
chosen from the strains Bacillus subtilis 1104, deposited as NRRL
B-67258; Bacillus subtilis 1781, deposited as NRRL B-67259;
Bacillus subtilis 747, deposited as NRRL B-67257; Bacillus subtilis
1541, deposited as NRRL B-67260; Bacillus subtilis 1999, deposited
as NRRL B-67318; and Bacillus subtilis 2018, deposited as NRRL
B-67261.
15. The composition of claim 13, wherein the at least one isolated
Bacillus strain is chosen from at least one of strains Bacillus 747
(NRRL B-67257) or a strain having all of the identifying
characteristics of Bacillus 747 (NRRL B-67257), Bacillus strain
1104 (NRRL B-67258) or a strain having all of the identifying
characteristics of Bacillus strain 1104 (NRRL B-67258), Bacillus
strain 1781 (NRRL B-67259) or a strain having all of the
identifying characteristics of Bacillus strain 1781 (NRRL B-67250),
Bacillus strain 1541 (NRRL B-67260) or a strain having all of the
identifying characteristics of Bacillus strain 1541 (NRRL B-67260),
Bacillus strain 1999 (NRRL B-67318) or a strain having all of the
identifying characteristics of Bacillus strain 1999 (NRRL B-67318);
and Bacillus strain 2018 (NRRL B-67261) or a strain having all of
the identifying characteristics of Bacillus strain 2018 (NRRL
B-67261).
16. The composition of claim 15, further comprising a carrier.
17. The composition of claim 16, wherein the carrier is selected
from a group consisting of: whey, maltodextrin, sucrose, dextrose,
limestone, rice hulls, and sodium silica aluminate.
18-35. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent
Application No. 62/219,433 filed Sep. 16, 2015; the entirety of
which is incorporated by reference herein.
BIBLIOGRAPHY
[0002] Complete bibliographic citations of those references that
are referred to herein by the first author's last name and year of
publication in parentheses can be found in the Bibliography
section, which precedes the claims.
FIELD OF THE INVENTION
[0003] This invention relates to compositions of novel
microorganisms for improving gastrointestinal homeostatis by
reducing bacterial pathogens, stimulating the host immune function
and thus reducing poultry diseases and enhancing health and
performance.
BACKGROUND OF THE INVENTION
[0004] Conventional poultry production uses antibiotics to prevent
disease and stimulate animal growth. Over time as a group of
animals is continually fed sub-therapeutic levels of antibiotics to
enhance their growth, susceptible bacteria within the
gastrointestinal tract of these animals will develop resistance.
When these bacteria are ingested via improperly handled meat it is
possible for individuals to become ill, and such individuals may
not respond to treatment with antibiotics that are the same or
similar to those fed to the animals. Therefore, it is recommended
that antibiotics used to treat human illnesses not be administered
to food animals. The World Health Organization (WHO) urges efforts
to phase out antimicrobials that are used to treat humans for
growth promotion in livestock (WHO Global Strategy
Recommendations). Motivated by health concerns over the potential
of antibiotic resistance bacteria in the food supply, environmental
concerns, animal welfare and quality concerns, many consumers are
seeking alternatives to conventional meat products that are
typically produced with routine use of antibiotics (Allen and
Stanton, 2014; Cheng et al., 2014). Accordingly, consumer demand
for chicken and turkey that has been raised without the use of
antibiotics is growing to the point that production of poultry
raised without the routine use of antibiotics has become part of
the mainstream.
[0005] Common bacterial disease challenges facing poultry that are
conventionally treated with antibiotics include colibacillosis
caused by Avian Pathogenic Escherichia coli (APEC), as well as
enteric diseases caused by various species from the Clostridium
genus.
[0006] Although Escherichia coli are normal residents of the
gastrointestinal tract in poultry, some strains carry virulence
genes and are able to cause colibacillosis in birds. These virulent
E. coli strains, known as Avian Pathogenic Escherichia coli (APEC),
are a heterogeneous group comprising a wide diversity of serotypes
and containing an array of virulence genes (Guabiraba and Schouler,
2015). Collibacillosis in poultry may be localized or systemic and
includes disease states such as colisepticemia, coligranuloma
(Hjarre's disease), air sac disease (chronic respiratory disease,
CRD), swollen-head syndrome, venereal colibacillosis and coliform
cellulitis (inflammatory process), peritonitis, salpingitis,
orchitis, osteomyelitis/synovitis (turkey osteomyelitis complex),
panophthalmitis, omphalitis/yolk sac infection and enteritis
(Barnes H J et al., 2008). Although difficult to quantify these
various disease forms are responsible for significant economic
losses in poultry. For instance, lesions consistent with
colisepticemia were present on 43% of broiler carcasses condemned
at processing. A reduction in the levels of APEC strains will
reduce rates of disease and have a positive effect on the
productivity of commercial broiler operations.
[0007] Necrotic enteritis, caused by C. perfringens, is the most
common and severe clostridial enteric disease in poultry (Barnes H
J, 2008; Cooper et al., 2013). Necrotic enteritis outbreaks are
sporadic, but typically occur in broilers between 2-6 weeks of age
(Cooper et al., 2013). It has been estimated that global necrotic
enteritis outbreaks result in a loss of over $2 billion annually
through increased medical costs, reduced weight gain and mortality
amongst animals (Lee et al., 2011a; Timbermont et al., 2011). The
characteristic intestinal lesions are generally considered to be
caused by the production of alpha toxin by C. perfringens Type A
(Al-Sheikhly and Truscott, 1977a, 1977b, 1977b) with NE toxin B
(NetB) also having been implicated in disease (Keyburn et al.,
2008, 2008, 2010a; Rood et al., 2016). C. perfringens is a normal
resident of the intestinal tract of poultry usually at levels below
10.sup.4 CFU/g intestinal contents, but found at levels about
10.sup.7 CFU/g in diseased birds (Shojadoost et al., 2012).
Therefore, maintaining low levels of C. perfringens can ameliorate
the onset of disease. Furthermore, C. perfringens infections have
been shown to increase when antibiotic growth promoters were
removed from poultry feed in Scandinavian countries, and it is
anticipated that the forthcoming removal of antibiotic growth
promoters from poultry feed in the USA will have a similar effect
(Grave et al., 2004; Immerseel et al., 2009; Kaldhusdal and
Lovland, 2000).
[0008] Bacteriocins, small antimicrobial peptides produced by
bacteria, are alternatives to common antibiotics in livestock
production. The function of bacteriocins is to allow the producer
cells to compete with other microbes in their natural environment.
They generally increase membrane permeability by forming pores in
membranes of target cells or inhibit cell wall synthesis thereby
preventing growth of susceptible microbes. Other beneficial
attributes of bacteriocins include resistance to low pH and heat
and little, if any, negative effects on host cells. These
bacterially produced antimicrobial peptides are very similar to
those produced by the host organism itself. Cationic antimicrobial
peptides, such as cathelicidins, are abundantly expressed in the
mucosal epithelial cells lining the digestive, respiratory and
reproductive tracts, as well as in the primary and secondary immune
organs of chickens, where they play an essential role in innate
defense and disease resistance (Achanta et al., 2012).
[0009] There may be concern that continual exposure of bacteria to
continual, high levels of bacteriocins could result in resistance
developing as it does for conventional antibiotics. This risk can
be greatly reduced by the combined use of a number of bacteriocins
with different mechanisms of action (Riley et al., 2012).
Synergistic effects between the bacteriocins allow for lower doses
and multiple spontaneous mutations will have to occur to acquire
resistance to a combination of bacteriocins.
[0010] What is needed are bacterial strains and combinations of
bacterial strains that are bacteriocin producing as to be useful in
poultry and other animals. Methods of making and using bacteriocin
producing bacterial strains and combinations thereof are also
needed. Additionally, methods of identifying bacteriocin producing
bacterial strains that are useful in poultry and other animals are
also needed.
SUMMARY OF THE INVENTION
[0011] The present invention, is intended to solve one or more of
the problems noted above.
[0012] In accordance with an embodiment of the present invention, a
composition comprising at least one isolated Bacillus strain chosen
from at least one of strains 747, 1104, 1781, 1541, and 2018 is
provided. The composition, including the at least one isolated
Bacillus strain may inhibit a pathogen chosen from at least one of
E. coli and Clostridium in an animal.
[0013] In accordance with another embodiment of the present
invention, the composition may comprise a plurality of isolated
Bacillus strains chosen from the strains 747, 1104, 1781, 1541, and
2018.
[0014] In accordance with another embodiment of the present
invention, the composition may comprise a plurality of isolated
Bacillus strains chosen from the strains 747, 1104, 1781, 1541,
1999 and 2018.
[0015] In accordance with another embodiment of the present
invention, the composition may further comprise a carrier selected
from a group consisting of but not limited to: whey, maltodextrin,
sucrose, dextrose, limestone, rice hulls, and sodium silica
aluminate. The carrier may be in the physical of a powdered solid,
a liquid, or a gel.
[0016] In accordance with another embodiment of the present
invention, the composition may also comprise an animal feed,
wherein the composition of the at least one isolated Bacillus
strain in said composition is about 1.times.10.sup.8 CFU/g.
[0017] In accordance with another embodiment of the present
invention, the composition may also comprise a liquid, such as
water, wherein the composition of the at least one isolated
Bacillus strain in said composition is about 1.times.10.sup.8
CFU/g.
[0018] In accordance with another embodiment of the present
invention, a combination is provided including one or more of
isolated Bacillus 747 (NRRL B-67257) or a strain having all of the
identifying characteristics of Bacillus 747 (NRRL B-67257),
Bacillus strain 1104 (NRRL B-67258) or a strain having all of the
identifying characteristics of Bacillus strain 1104 (NRRL B-67258),
Bacillus strain 1781 (NRRL B-67259) or a strain having all of the
identifying characteristics of Bacillus strain 1781 (NRRL B-67250),
Bacillus strain 1541 (NRRL B-67260) or a strain having all of the
identifying characteristics of Bacillus strain 1541 (NRRL B-67260),
and Bacillus strain 2018 (NRRL B-67261), Bacillus strain 1999 (NRRL
B-67318) or a strain having all of the identifying characteristics
of Bacillus strain 1999 (NRRL B-67318); and Bacillus strain 2018
(NRRL B-67261), or a strain having all of the identifying
characteristics of Bacillus strain 2018 (NRRL B-67261).
[0019] The Bacillus strains identified herein according to one
embodiment of the present invention, to inhibit pathogens, produce
multiple compounds with inhibitory activity against other microbes
with many strains containing more than ten operons producing
antifungal and antibacterial compounds. Multiple bacteriocins are
being produced in vitro directly at the site of action by the
Bacillus strains so a robust blend of bacteriocins are present at
doses lower than would be needed if isolated bacteriocins were
being added directly to the feed.
[0020] Both in vitro data and in vivo trials indicate the
effectiveness of these Bacillus strains in inhibiting poultry
pathogens, such as APEC and C. perfringens, thereby decreasing the
disease-burden in commercial broiler operations.
[0021] Accordingly, in accordance with another embodiment of the
present invention, a method is provided comprising administering to
an animal an effective amount of at least one isolated Bacillus
strain chosen from the strains 747, 1104, 1781, 1541, and 2018 to
inhibit a pathogen chosen from at least one of E. coli and
Clostridium in the animal.
[0022] In accordance with another embodiment of the present
invention, the animal may be a chicken or a turkey.
[0023] In accordance with another embodiment of the present
invention, administering said strain or strains to said animal
improves average daily weight gain relative to that in animals that
have not been administered the strain or strains.
[0024] In accordance with another embodiment of the present
invention, administering said strain or strains to a first group of
said animals decreases mortality rate amongst the group of animals
relative to that in second group of animals that have not been
administered the strain or strains.
[0025] In accordance with another embodiment of the present
invention, administering said strain or strains to said animal
reduces a level of C. perfringens Type A in gastrointestinal tract
tissue of the animal relative to that in animals that have not been
administered the strain or strains.
[0026] In accordance with another embodiment of the present
invention, the level of C. perfringens Type A in the treated animal
is reduced by about 85.0% relative to that in animals that have not
been administered the strain or strains.
[0027] In accordance with another embodiment of the present
invention, the level of C. perfringens Type A in gastrointestinal
tract tissue of the treated animal is less than about 50 CFU/g.
[0028] In accordance with another embodiment of the present
invention, the occurrence of necrotic enteritis in the treated
animal is reduced relative to that in animals that have not been
administered the strain or strains according to the present
invention.
[0029] In accordance with another embodiment of the present
invention, administering said Bacillus strain or strains to said
animal reduces a level of avian pathogenic E. coli (APEC) in
gastrointestinal tract tissue of the animal relative to that in
animals that have not been administered the strain or strains.
[0030] In accordance with another embodiment of the present
invention, the level of avian pathogenic E. coli (APEC) in the
treated animal is reduced by about 80.0%.
[0031] In accordance with another embodiment of the present
invention, the occurrence of colibacillosis in the treated animal
is reduced relative to that in animals that have not been
administered the strain or strains.
[0032] In accordance with another embodiment of the present
invention, the administering an effective amount of a plurality of
isolated Bacillus strains increases a concentration of a plurality
of bacteriocins in the gastrointestinal tract tissue of the
animal.
[0033] In accordance with another embodiment of the present
invention, the effective amount of plurality of isolated Bacillus
strains are administered to the animal in the form of a direct fed
microbial composition including a comprises a carrier.
[0034] In accordance with another embodiment of the present
invention, administering an effective amount of a plurality of
isolated Bacillus strains to the animal modulates the immune system
of the treated animal.
[0035] In accordance with another embodiment of the present
invention, the plurality of isolated Bacillus strains are chosen
from the strains 747, 1104, 1781, 1541, 1999 and 2018.
BRIEF DESCRIPTION OF THE DRAWING
[0036] FIG. 1: Levels (CFU/g) of APEC in broiler GITs from
untreated birds and birds treated with a direct fed microbial
product according to one embodiment of the present invention. Black
lines indicate mean with SEM. Superscripts of different letters
denote significance (P<0.05 by unpaired, two-tailed t-test);
[0037] FIG. 2: Levels (CFU/g) of C. perfringens in broiler GITs
from untreated birds and birds treated with a direct fed microbial
product according to one embodiment of the present invention. Black
lines indicate mean with SEM. Superscripts of different letters
denote significance (P<0.05 by unpaired, two-tailed t-test);
[0038] FIG. 3: Levels (CFU/g) of Clostridium perfringens in turkey
GITs from untreated birds and birds treated with a direct fed
microbial product according to one embodiment of the present
invention. Black lines indicate mean with SEM. Superscripts of
different letters denote significance (P<0.05 by unpaired,
two-tailed t-test);
[0039] FIG. 4. Whole genome phylogenetic tree indicating the
relatedness of strains to each other. Bacillus strains according to
the present invention are identified in dark gray and previously
identified strains are identified in light gray;
[0040] FIG. 5: Levels (CFU/g) of APEC in turkey GITs from untreated
birds (sampling 1) and birds treated with a direct fed microbial
product according to one embodiment of the present invention
(samplings 2 & 3). Black lines indicate mean with SEM.
Superscripts of different letters denote significance (P<0.05 by
one-way ANOVA multiple comparison analysis).
[0041] FIG. 6: Levels (CFU/g) of APEC in broiler GITs from
untreated birds (samplings 1, 2 and 4) and birds treated with a
direct fed microbial product according to one embodiment of the
present invention (sampling 3). Superscript with different letters
denote significance (P<0.05 by one-way ANOVA multiple comparison
analysis);
[0042] FIG. 7: Levels (CFU/g) of Clostridium perfringens in broiler
GITs from untreated birds (samplings 1, 2 and 4) and birds treated
with a direct fed microbial product according to one embodiment of
the present invention (sampling 3). Superscript with different
letters denote significance (P<0.05 by one-way ANOVA multiple
comparison analysis);
[0043] FIG. 8: Average body weight of broilers at 14 days of age.
Chickens were fed basal diets (CON), diets supplemented with
antibiotic (BMD) or various strains of Bacillus according to the
present invention (PB1, PB2, PB3). The data were analyzed using
one-way ANOVA and the means were separated using Duncan's multiple
range test. The asterisk (*) denotes significantly increased body
weights compared with controls (P<0.05);
[0044] FIG. 9: Average FCR of broilers at 14 days of age. Chickens
were fed either basal diets (CON), diets supplemented with
antibiotic (BMD) or various strains of B. subtilis (PB1, PB2, PB3).
The data were analyzed using one-way ANOVA and the means were
separated using Duncan's multiple range test. The asterisk (*)
denotes significantly increased FCR compared with controls
(P<0.05);
[0045] FIG. 10: Effects of dietary direct fed microbial or
antibiotics on the levels of pro-inflammatory cytokine transcripts:
A. IL1.beta., B. IL6, C. IL8, D. IL17F and E. TNFSF15. Chickens
were fed either basal diets (CON), diets supplemented with
antibiotic (BMD) or various strains of Bacillus according to the
present invention (PB1, PB2, PB3). Transcript levels of various
cytokines in the ileum were measured using quantitative RT-PCR and
normalized to GAPDH transcript levels. The data were analyzed using
one-way ANOVA and the means were separated using Duncan's multiple
range test. Each bar represents the mean.+-.SEM (n=6). The asterisk
(*) denotes significantly increased expression compared with
controls (P<0.05);
[0046] FIG. 11: Effects of dietary direct fed microbial or
antibiotics on the levels of transcripts of Th1 (A. IL2, B.
IFN.gamma.), Th2 (C. IL4, D. IL13) and regulatory cytokines (E.
IL10). Chickens were fed either basal diets (CON), diets
supplemented with antibiotic (BMD) or various strains of Bacillus
according to the present invention (PB1, PB2, PB3). Transcript
levels of various cytokines in the ileum were measured using
quantitative RT-PCR and normalized to GAPDH transcript levels. The
data were analyzed using one-way ANOVA and the means were separated
using Duncan's multiple range test. Each bar represents the
mean.+-.SEM (n=6). The asterisk (*) denotes significantly increased
expression compared with controls (P<0.05); and
[0047] FIG. 12: Effects of dietary direct fed microbial or
antibiotics on the levels of transcripts of TJ proteins (A. JAM2,
B. occludin, C. ZO1) and mucin (D. MUC2). Chickens were fed either
basal diets (CON), diets supplemented with antibiotic (BMD) or
various strains of Bacillus according to the present invention
(PB1, PB2, PB3). Transcript levels of various TJ proteins and mucin
in the ileum were measured using quantitative RT-PCR and normalized
to GAPDH transcript levels. The data were analyzed using one-way
ANOVA and the means were separated using Duncan's multiple range
test. Each bar represents the mean.+-.SEM (n=6). The asterisk (*)
denotes significantly increased expression compared with controls
(P<0.05).
[0048] Before explaining embodiments of the invention in detail, it
is to be understood that the invention is not limited in its
application to the details of construction and the arrangement of
the components set forth in the following description or
illustrated in the drawings. The invention is capable of other
embodiments or being practiced or carried out in various ways.
Also, it is to be understood that the phraseology and terminology
employed herein is for the purpose of description and should not be
regarded as limiting.
DETAILED DESCRIPTION
[0049] In accordance with the present invention, there may be
employed conventional molecular biology and microbiology within the
skill of the art. Such techniques are explained fully in the
literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular
Cloning: A Laboratory Manual, Third Edition (2001) Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y.
[0050] Bacterial strains useful for improving health and
performance of poultry are provided in accordance with the present
invention. In one embodiment of the invention, bacteria strains
belong to the genus Bacillus. One or more Bacillus strains can be
used in combination. The Bacillus strain(s) can be fed to poultry
as a direct-fed microbial (DFM), dosed through the drinking water
line or applied using a spray cabinet on newly hatched birds at the
hatchery. Feeding or dosing one or more Bacillus strains described
herein reduces bacterial pathogens of poultry, improves immune and
gut barrier function and GI microbial homeostasis resulting in
improved health and performance in poultry.
[0051] Bacillus strains--isolation and characterization
[0052] Bacillus strains described herein were isolated from
environmental sources including water, animal feed, fermented
silages, poultry litter and soil. Samples were heat shocked to kill
the vegetative bacterial populations and inoculated on general
media to grow out the spore-forming bacteria into colonies. Plates
were incubated at mesosphilic temperatures under aerobic conditions
to inhibit the growth of anaerobic bacteria. Representative
colonies were picked, grown in broth overnight and the resulting
cell mass harvested and split for long-term storage and DNA
isolation. Genomic DNA was harvested from each strain and used as a
template for PCR of the 16S rRNA gene for strain identification and
RAPD analysis to determine the relatedness among the strains.
Strains identified as belonging to Bacillus species Generally
Recognized As Safe (GRAS) were further tested for safety and
selected based on functional pathogen inhibition assays.
[0053] Bacillus strains--selection
[0054] Bacillus strains were selected as candidates for use as
Direct Fed Microbials (DFM) based on functional inhibitory assays
against avian pathogenic E. coli and Clostridium perfringens.
Alternatively, strains were selected using genetic screening
techniques to identify strains with targeted antimicrobial genes.
Both techniques were successful at identifying candidate Bacillus
strains for DFM.
[0055] For functional testing, APEC and C. perfringens strains were
isolated from poultry flocks and used as indicators in
antimicrobial broth assays. Bacillus strains were grown overnight,
cells removed by centrifugation and cell-free supernatants were
prepared by filter sterilization. Antimicrobial assays contained
indicator strains inoculated in growth medium and mixed with
Bacillus supernatants. Assays were incubated overnight and the
growth compared to assays of the same indicator strain without the
Bacillus supernatants. The most effective inhibitory strains
showing the highest growth inhibition against the broadest
collection of APEC and C. perfringens indicator strains were
identified and prepared for scale-up production.
[0056] Alternatively, Bacillus strains were selected based on
presence of antimicrobial genes using genetic screening methods.
Genes with known inhibitory activity were identified and primer
sequences constructed. PCR reactions with genomic DNA from Bacillus
strains and the constructed primers were used to identify strains
with the antimicrobial gene of interest. Bacillus strains shown to
produce the known PCR amplicon were chosen for functional
antimicrobial screening. Strains confirmed to have the known
activity were prepared for large-scale production.
[0057] Bacillus strains--beneficial activities
[0058] Bacillus strains have a number of activities that make them
efficacious for feeding poultry including the production of
extracellular enzymes, antimicrobials and immune modulating
molecules. In addition, Bacillus form endospores that make them
stable in feed and other feed components. These spores are heat
resistant and thus will survive normal feed pelleting processes.
The spores are recalcitrant to drying and mineral salts making them
stable in vitamin and trace mineral premixes.
[0059] The Bacillus strains described herein produce a number of
different antimicrobials such as polyketides and lipopeptides as
well as larger protein bacteriocin-like molecules that effectively
inhibit enteric disease causing clostridia including C. perfringens
and C. septicum. In addition, some of these of these antimicrobials
are effective at inhibiting APEC isolates. Combining multiple
Bacillus strains can effectively produce a DFM product for
broad-spectrum control of important disease-causing bacteria in
poultry.
[0060] In addition to the antimicrobial activities, Bacillus
produce a number of extracellular enzymes including cellulase,
hemicellulase, xylanase, amylase and proteases. These exogenous
enzymes play a role in improving the utilization of some of the
difficult to digest feed components such as non-starch
polysaccharides which can have a negative impact on feed efficiency
in poultry. These enzymes also alter the nutrient levels in the GI
tract such as decreasing starch levels in the lower GI tract, which
reduces the potential of proliferation of starch utilizing
clostridia. Thus, the Bacillus enzyme activity plays a role in
maintaining microbial gut homeostasis.
[0061] Other activities of the Bacillus that are important to
improve poultry performance include the production of immune
modulating molecules. Many factors such as diet changes, disease
challenges and stress can affect gut health. These factors lead to
the loss of structural integrity of the intestinal epithelium
resulting in a decrease in the absorptive surface, increase in
intestinal permeability and increase in inflammatory responses that
ultimately reduce performance. Increased permeability directly
results in the translocation of bacteria and their metabolic
products into circulation. Feeding selected strains of Bacillus has
been shown to alter intestinal immune activity and improve gut
barrier integrity through increased expression of tight junction
(TJ) proteins. Increased TJ protein expression in chickens fed
Bacillus-supplemented diets translates to increased intestinal
barrier function and optimal gut health.
[0062] Bacillus strains identified as being useful and containing
one of more of these beneficial activities include strains 747,
1104, 1541, 1781, 1999 and 2018. These strains can be fed
individually or in combination with each other.
[0063] Bacillus strains 747, 1104, 1541, 1781 and 2018 were
deposited on May 24, 2016 at the Agricultural Research Service
Culture Collection (NRRL), 1815 North University Street, Peoria,
Ill., 61604 and given accession numbers NRRL B-67257 for strain
747, NRRL B-67258 for strain 1104, NRRL B-67260 for strain 1541,
NRRL B-67259 for strain 1781 and NRRL B-67261 for strain 2018.
Strain 1999 was deposited on Sep. 15, 2016 and given the accession
number NRRL B-67318. All deposits were made under the provisions of
the Budapest Treaty on the International Recognition of the Deposit
of Microorganisms for the Purposes of Patent Procedure.
[0064] Bacillus as Direct-Fed Microbials
[0065] Administration of one or more Bacillus microorganisms to
poultry may be accomplished by several methods including adding the
Bacillus strains to the animals' feed, or drinking water, or to the
bedding or litter, or by spraying on the chicks or poults at
hatching such as by an aerosol or gels. Bacillus strains according
to the present invention can be administered as a direct-fed
microbial concentrate which is mixed into the feed as part of the
vitamin mineral premix or as a separate inclusion into the feed or
as a water soluble concentrate that is added to the drinking water
system via a proportioner and diluted into the nipple drinkers. In
addition, Bacillus strains can be administered to a newly hatched
chick or poult via a spray aersol immediately after hatching and
before placement on the farm.
[0066] Bacillus strains may be administered in various forms, for
example as a feed supplement via the vitamin trace mineral premix
or as a separate concentrate for mixing into the feed. In one
embodiment of the feed supplement form, freeze-dried Bacillus
fermentation product in the form of spores is added to a carrier,
such as whey, maltodextrin, sucrose, dextrose, limestone (calcium
carbonate), rice hulls, sodium silica aluminate. In one embodiment
of the liquid drench, freeze-dried Bacillus spores product is added
to a carrier, such as maltodextrin, sucrose, dextrose, dried
starch, sodium silica aluminate, and a liquid used in a spray
cabinet. In one embodiment of the gel form, freeze-dried Bacillus
fermentation product is added to a carrier, such as starch or other
or carbohydrates based gums, sucrose, silicon dioxide, polysorbate
80, propylene glycol and artificial coloring to form the gel.
[0067] The Bacillus strains are grown in a liquid broth containing
protein, carbohydrates and minerals at a constant temperature and
agitation to maximize the initial cell density. In the initial
phase of the fermentation, the conditions are set to maximize the
cell density and then in the later stages of the fermentation
conditions are set to convert the cells to spores. In one
embodiment, the strains are grown to an initial OD in Nutrient
broth where the cell yield is at least 2.times.10.sup.9 colony
forming units (CFU) per ml of culture. Following the initial growth
phase, agitation can be reduced, supplements added to induce
sporulation and the cells convert to spore forms. Once the culture
reaches a maximum spore density, the culture is harvested by
separating the cells from the medium by centrifugation. Wet spore
paste is then mixed with stabilizing agents such as starch,
maltodextrin, citric acid and cryoprotectants if the paste is to be
freeze-dried. The suspended spore paste is then dried and milled to
provide a flowable powder.
[0068] To prepare compositions, the dried spore powder can be added
to a carrier such as whey, maltodextrin, sucrose, dextrose,
limestone (calcium carbonate), rice hulls, sodium silica aluminate
in a ribbon or paddle mixer and mixed to produce an even
distribution of the spores in the carrier. The components are
blended such that a uniform mixture of the carrier and cultures
result.
[0069] A preferred dosage for the premix or concentrate product is
about 1.times.10.sup.7 CFU/g to about 1.times.10.sup.9 CFU/g, and
more preferably about 1.times.10.sup.8 CFU/g. One pound of the
concentrate or premix is then added to a finished ton of feed to
provide 1.5.times.10.sup.5 cfu/g of feed. A preferred dosage range
for inclusion into water is about 1.times.10.sup.6 CFU/g to about
1.times.10.sup.8 CFU/g, and more preferably about 1.times.10.sup.7
CFU/g. A preferred dosage range of the liquid drench and gel is
about 1.times.10.sup.6 CFU/g to about 1.times.10.sup.9 CFU/g, and
more preferably about 1.times.10.sup.8 CFU/g.
[0070] While these above listed examples of the present invention
disclose the use of freeze-dried Bacillus as an ingredient for the
premix, concentrate, gel or water form, the present invention is
not limited to use of freeze-drying Bacillus before administrating
to poultry, and non-freeze dried Bacillus are considered well
within the scope of the present invention. For example,
spray-dried, fluidized bed dried, or solid-state fermentation or
Bacillus in other states may be used in accordance with the present
invention.
[0071] When fed to an animal, such as poultry, Bacillus become
established within the animal's gastrointestinal tract. As a result
of the Bacillus strains, 747, 1104, 1541, 1781, and 2018 of the
present invention becoming established in the animal's
gastrointestinal tract, a significant reduction in pathogen load
for both C. perfringens and APEC may be obtained.
[0072] Also described herein is a method of reducing both C.
perfringens and APEC pathogen load in broiler chickens by
administering a DFM including one or more Bacillus strains, 1104,
1541, and 1781. In this method, samples of gastrointestinal tracts
(GIT) of broiler chicken populations which had and had not been
administered with a DFM including one or more Bacillus strains,
1104, 1541, and 1781 were sampled. According to this method
portions of gastrointestinal tracts of broilers are obtained and E.
coli colonies are counted and recorded. A selection of isolated E.
coli colonies is made and the genomic DNA of the selected E. coli
colonies is extracted. The DNA is amplified and APEC pathotype is
determined using multiplex polymerase chain reaction (PCR). To
verify that the E. coli is APEC, the PCR product is then run
through capillary gel electrophoresis and each isolate is confirmed
to contain at least two of the five following APEC-associated
virulence genes in its genome: hlyF, ompT, iroN, iss, iutA.
Similarly, portions of gastrointestinal tracts of broilers is
obtained and C. perfringens colonies are counted and record. A
selection of isolated C. perfringens colonies is made and the
genomic DNA of the selected C. perfringens colonies is extracted.
The C. perfringens DNA is amplified and C. perfringens toxinotype
is determined using multiplex polymerase chain reaction (PCR) to
amplify the alpha toxin gene. In order to eliminate the
non-perfringens Clostridium species the PCR product is then run
through capillary gel electrophoresis and each isolate is confirmed
to be C. perfringens. According to this method, pathogen load for
APEC in broilers treated with the DFM containing the Bacillus
strains, 1104, 1541, and 1781, is on average 2.1.times.10.sup.4
CFU/g as compared to untreated broilers, which yield an average
APEC level of 2.1.times.10.sup.5 CFU/g. Similarly, according to
this method, pathogen load for C. perfringens in broilers treated
with the DFM containing the Bacillus strains, 1104, 1541, and 1781,
is on average 50 CFU/g as compared to untreated broilers, which
yield an average C. perfringens level of 1.7.times.10.sup.2
CFU/g.
[0073] Also described herein is a method of reducing both C.
perfringens pathogen load in turkeys by administering a DFM
including Bacillus strain 1104 in combination with commercially
available strain Bs2084(Microbial Discovery Group, Franlkin, Wis.).
In this method, samples of gastrointestinal tracts (GIT) of turkey
populations which had and had not been administered with a DFM
including Bacillus strain 1104 in combination with commercially
available strain Bs2084 (Microbial Discovery Group, Franlkin, Wis.)
were collected. According to this method portions of
gastrointestinal tracts of turkeys are obtained and C. perfringens
colonies are counted and record. A selection of isolated C.
perfringens colonies is made and the genomic DNA of the selected C.
perfringens colonies is extracted. The C. perfringens DNA is
amplified and C. perfringens toxinotype is determined using
multiplex polymerase chain reaction (PCR) to amplify the alpha
toxin gene. In order to eliminate the non-perfringens Clostridium
species the PCR product is then run through capillary gel
electrophoresis and each isolate is confirmed to be C. perfringens.
According to this method, pathogen load for C. perfringens in
turkeys treated with the DFM containing the including Bacillus
strain 1104 in combination with commercially available strain
Bs2084 (Microbial Discovery Group, Franlkin, Wis.)is on average 50
CFU/g as compared to untreated turkeys, which yield an average C.
perfringens level of 1.7.times.10.sup.2 CFU/g.
[0074] While these examples use freeze-dried Bacillus as an
ingredient for the premix, concentrate, gel or water form, it is
not necessary to freeze-dry the Bacillus before administrating to
poultry. For example, spray-dried, fluidized bed dried, or
solid-state fermentation or Bacillus in other states may be
used.
EXAMPLES
[0075] The following Examples are provided for illustrative purpose
only. The Examples are included herein solely to aid in a more
complete understanding of the presently described invention. The
Examples do not limit the scope of the invention described or
claimed herein in any fashion.
Example 1
Isolation and Selection of Bacillus Strains 747, 1104, 1541, 1781
and 2018
[0076] Design. Select Bacillus isolates for their antimicrobial
properties against avian pathogens Clostridium perfringens and
avian pathogenic Escherichia coli (APEC) as candidate strains for
use as a DFM to be applied in the poultry industry. Aerobic
sporeforming bacteria were isolated from a variety of environmental
sources and characterized using RAPD-PCR. Genetically-similar
groups (184 representatives) were identified by sequencing the 16S
rRNA gene and strains that were recognized to be non-GRAS species
were eliminated. The remaining strains were then screened by
exposing their cell-free bacteriocin to a genetically diverse panel
of C. perfringens and APEC isolates to test for inhibition. After
confirming the absence of the Bacillus cereus emetic toxin, the
isolates with the greatest inhibition potential were selected to be
commercialized.
[0077] Materials and Methods. Isolation of spore forming strains:
117 samples from various environmental sources were diluted with 99
mL of sterile 0.1% peptone broth and spore-treated in a 65.degree.
C. water bath for 30 min. Serial dilutions were made and
pour-plated with tempered molten TSA (Becton, Dickenson &
Company, Franklin Lakes, N.J.) and incubated at 32.degree. C. for
12-24 h. Several isolated colonies from each sample were picked and
struck to TSA plates for isolation and incubated at 32.degree. C.
for 12-24 h. Isolated colonies were picked and used to inoculate 3
mL TSB (Becton, Dickenson & Company, Franklin Lakes, N.J.) in a
well of a 12-well culture plate (Falcon, Tewksbury, Mass.) and
incubated at 32.degree. C., shaking 100-125 rpm for 12-24 h. The
growth culture was spun down and resuspended in 2 mL TSB with 20%
glycerol. 1 mL of this mixture was frozen at -20.degree. C. to be
used for gDNA isolation, while the remaining 1 mL was frozen at
-80.degree. C. as frozen cell stock.
[0078] DNA Isolation: Genomic DNA was isolated from all 2029
isolated strains using either the Roche Applied Science High Pure
PCR Template Kit or the following DNA isolation protocol: Add 20
.mu.L of lysozyme (100 mg/mL) to 300 .mu.L of overnight growth in
TSB and incubate at 37.degree. C. for 60 min, add 220 .mu.L of
lysis buffer (6 M Guanidine, 20% Triton-X 100, 10 mM Tris-HCl, pH
7.5) and incubate at 25.degree. C. for 15 min, add 20 .mu.of
Protease K 800 U/ml (NEB, Ipswich, Mass.) and incubate at
55.degree. for 30 min, transfer 400 .mu.L of lysate to a
Wizard.RTM. SV 96 Binding Plate (Promega, Fitchburg, Wis.) and
continue with manufacturer's filtration instructions from
Wizard.RTM. SV 96 Genomic DNA Purification System starting from
step 3.C.4 (4/15/revision) (Promega, Fitchburg, Wis.).
[0079] RAPD-PCR Profiles: All isolated strains were subjected to
RAPD-PCR in order to generate strain-specific RAPD profiles. Each
reaction contained 1 Ready-To-Go RAPD Analysis Bead (GE Healthcare,
Chicago, Ill.), 2.5 .mu.L RAPD primer 3 (5'-d{GTAGACCCGT}-3',
(Eurofins, Brussels, Belgium), 5 .mu.L template gDNA and 17.5 .mu.L
nuclease-free water. Incubations were executed using an Applied
Biosystems Veriti.RTM. Thermal Cycler (ThermoFisher Scientific,
Milwaukee, Wis.) with the following protocol: 1 cycle at 95.degree.
C. for 5 minutes, 45 cycles of 95.degree. C. for 1 minute,
36.degree. C. for 1 minute, and 72.degree. C. for 2 minutes. The
PCR product was run through capillary gel electrophoresis using a
Fragment Analyzer.TM. from Advance Analytical Technologies, Inc.
and visualized on PROsize 2.0 (Advanced Analytical Technologies,
Inc., Ames, Iowa). RAPD profiles were imported into BioNumerics
(Applied Maths, Sint-Martens-Latem, Belgium) and a dendrogram was
generated using Dice and the UPGMA algorithm method. Clusters were
established using a 75% similarity cutoff and n representatives
were chosen from each cluster where n=the square root of the number
of cluster members.
[0080] 16S rRNA Sequencing: 408 representative strains were
subjected to 16 rRNA sequencing after amplification of the 16S rRNA
gene using primers 27F-YM (5'-d{AGAGTTTGATYMTGGCTCAG}-3',
(Eurofins, Brussels, Belgium) and 1492R-Y
(5'-d{TACCTTGTTAYGACTT}-3', (Eurofins, Brussels, Belgium). Strains
identified as GRAS Bacillus species allowed to proceed as candidate
Bacillus strains.
[0081] Indicator Pathogens: APEC and Clostridium perfringens Type A
isolates isolated from poultry were characterized based on a
RAPD-PCR (RAPD primer 2 [5'-d{GTTTCGCTCC}-3']). Genetically-varied
representative APEC and C. perfringens Type A isolates were
selected as indicators strains for the bacteriocin assay.
[0082] Bacteriocin Assay: Bacteriocin from 171 candidate Bacillus
strains was harvested from 3 mL of 18 h growth in TSB (30.degree.
C., shaking 100 rpm) and sterilized by filtration with a 0.22 .mu.m
filter (Merck Millipore, Billerica, Mass.). For C. perfringens
assay: 70 .mu.L of cell-free bacteriocin was added to 600 .mu.L of
indicator strain (1% inoculum) and incubated at 37.degree. C.,
AnaeroPack System (Mitsubishi, New York, N.Y.), 12-18 h. Top
performers were subjected to the APEC assay: 200 .mu.L of cell-free
bacteriocin was added to 466 .mu.L of indicator strain (1%
inoculum) or 70 .mu.L of cell-free bacteriocin was added to 600
.mu.L of indicator strain (1% inoculum) and incubated at 37.degree.
C., shaking 100 rpm, 12-18 h.
[0083] Emetic Toxin screening: The five best performers were
screened for the Tecra.TM. Bacillus cereus emetic toxin using the
(3M, Maplewood, Minn.) Bacillus Diarrhoeal Enterotoxin Visual
Immunoassay kit.
[0084] Results and Discussion. 2029 aerobic sporeforming bacteria
were isolated from 117 environmental samples of various sources.
Each isolate was subjected to RAPD-PCR (RAPD primer 3) to produce
strain-specific fingerprints and dendrogram was generated based on
RAPD-type similarity. 408 isolates were selected as representatives
of 184 clusters and identified by sequencing the 16S rRNA gene. Of
these 171 isolates were identified as species on the GRAS list.
Cell-free bacteriocin of these 171 isolates was collected and
tested for inhibition against a genetically-varied panel of 15
Clostridium perfringens isolates and 16 APEC isolates. The top 5
performers were confirmed to be negative for the Bacillus cereus
emetic toxin and chosen as strains for commercial scale-up. These 5
strains were 747, 1104, 1541, 1781, and 2018.
Example 2
Isolation and Selection of Bacillus Strain 1999
[0085] Introduction. A detailed analysis of Bacillus genomes
indicated that gene clusters associated with production of
antimicrobials were present in one or more strains, but absent in
others. Genes for nonribosomal synthesized lipopeptides,
cyclodepsipeptides, post-translationally modified peptides and
polyketides were among those detected. None of the Bacillus
strains, according to the present invention, contained the
post-translationally modified peptide, plantazolocin, or the
uncharacterized NRPS gene cluster, both present in the
non-proprietary strain 2084. Primers were therefore developed to
detect the nrsF gene of the NRPD gene cluster and to the ycaO gene
of the plantazolicin operon.
[0086] Materials and Methods. PCR Amplification: Three primer sets
were developed for the ycaO gene of the plantazolicin operon and 2
primer sets for the nrsF gene of the NRPD gene cluster as shown
below in Table 1.
TABLE-US-00001 TABLE 1 Primers designed to detect the antimicrobial
genes unique to Bacillus strain 2084. Forward Reverse Amplicon
Primer Primer Primer Primer Size Gene Name (5'-3') Name (5'-3')
(bp) 2084 YcaO_ ACCAACATCATT YcaO_ GACGATATCGG 292 ycaO F1 GCGGCTAC
R1 TTCCTGCGT 2084 YcaO_ ACCTTTGTAGAA YcaO_ CACATCAATCT 102 ycaO F2
GCAGCAATTTCA R2 GGGGCAAGC 2084 YcaO_ TCATACGGAATG YcaO_
TCATATCAACTA 248 ycaO F3 GCCTGGGG R3 AGTGTAGCCGCA 2084 NrsF_
ACTTTTGTTGAA NrsF_ AGACGTTACGT 123 nrs F1 GTTGGCCCG R1 TTTCCCCCT
2084 NrsF_ ACAGTTGCTGTT NrsF_ CGGGCCAACTT 138 nrs F2 AGTGTCCCA R2
CAACAAAAG
[0087] PCR reactions were set up in 20 .mu.L volumes containing 2.0
.mu.L 10.times.PCR Buffer, 0.6 50 .mu.L mM MgCl.sub.2, 0.4 .mu.L
dNTPs (10 mM each), 2.8 .mu.L of each forward and reverse primer
(10 .mu.M each), 0.08 .mu.L Platinum Taq (Life Technologies
10966083) and 9.32 .mu.L ddH.sub.2O. Conditions were optimized for
the YcaO_F3/R3 and NrsF_F2/R2 primer sets and started with a 4
minute denaturation at 95.degree. C. followed by 30 cycles of
denaturation at 95.degree. C. for 30 seconds, annealing at
60.degree. C. for 30 seconds and extension at 72.degree. C. for 30
seconds before a final extension at 72.degree. C. for 7
minutes.
[0088] Bacteriocin Assay: Bacillus colonies were inoculated into 25
mL BHI in a 125 mL Erlenmeyer flask and incubated at 32.degree. C.
for 24 h with shaking (150 rpm). One mL was used to inoculate 100
mL BHI in a 500 mL Erlenmeyer flask and incubated at 32.degree. C.
for 24 h with shaking (150 rpm). The culture was split between two
50 mL conical tubes and centrifuged at 14,000.times.g for 20 min.
The supernatant was then filtered through a 0.2 .mu.m filter and
stored at -20.degree. C. before use.
[0089] The E. coli isolates were picked in to 10 mL of TSB and
incubated at 37.degree. C. for 24 h. 0.1 mL was then transferred to
10 mL TSB and incubated at 37.degree. C. for 6 hours. Again, 0.1 mL
of culture was used to inoculate 10 mL TSB and 600 .mu.L was
transferred into a 24 well culture plate with 70 .mu.L of
bacteriocin. The plates were incubated at 37.degree. C., with
shaking (150 rpm) for 16-20 h before ODs were taken to measure
inhibition.
[0090] Results. Genetic screening for new proprietary candidate DFM
strains: The inventor's library of over 2000 environmental
spore-forming bacteria was screened using the primers developed to
detect the nrsF gene of the NRPD gene cluster and to the ycaO gene
of the plantazolicin operon. Eighteen strains were positive for
both genes, nine for the ycaO gene only and two for the nrsF gene
only. Sequencing of the 16S rRNA gene indicated that all 29
belonged to the Bacillus subtilis group.
[0091] Functional screening for new product development: Twelve of
the 29 strains selected through genetic screening were tested in a
bacteriocin assay to inhibit a panel of 12 E. coli. Three strains
inhibited the panel of E. coli greater than 75%, two clustered with
the commercial strains with average inhibitions ranging from 50 to
75% and the remaining seven inhibited the panel less than 50% as
shown below in Table 2.
TABLE-US-00002 TABLE 2 Strains were selected based on the presence
of one or both antimicrobial associated genes, nrsF and ycaO from
2084. A subset was screened for bacteriocin activity against a
panel of 12 E. coli and were grouped according to inhibition.
Average 16S rRNA Sequence Strain Lab# nrsF ycaO Inhibition ID 1999
11.10.14 + + 89.4 Bacillus subtilis group 1145 6.5.14 + + 78.1
Bacillus subtilis group 967 5.5.2 + + 76.5 Bacillus subtilis group
2018 Commercial - - 61.6 Bacillus subtilis group 466 2.16.12 - +
59.0 Bacillus subtilis group 2084 Commercial + + 55.8 Bacillus
subtilis group 747 Commercial - - 54.4 Bacillus subtilis group 1382
8.5.2 - + 53.2 Bacillus subtilis group 1192 6.9.7 + + 48.3 Bacillus
subtilis group 1161 6.7.5 + + 47.5 Bacillus subtilis group 358
2.10.2 - + 46.1 Bacillus subtilis group 1879 10.12.22 + - 38.8
Bacillus subtilis group 1621 9.9.8 - + 23.3 Bacillus subtilis group
1073 6.1.8 + + 5.7 Bacillus subtilis group 1166 6.7.10 + + 0.3
Bacillus subtilis group 1201 6.9.16 + + Not Tested Bacillus
subtilis group 1169 6.7.13 + + Not Tested Bacillus subtilis group
1235 6.11.9 + + Not Tested Bacillus subtilis group 1240 6.11.14 + +
Not Tested Bacillus subtilis group 1072 6.1.7 + + Not Tested
Bacillus subtilis group 1067 6.1.2 + + Not Tested Bacillus subtilis
group 996 5.5.31 + + Not Tested Bacillus subtilis group 987 5.5.22
+ + Not Tested Bacillus subtilis group 839 5.1.34 + + Not Tested
Bacillus subtilis group 1952 11.7.9 + + Not Tested Bacillus
subtilis group 387 2.11.22 - + Not Tested Bacillus subtilis group
363 2.10.7 - + Not Tested Bacillus subtilis group 357 2.10.1 - +
Not Tested Bacillus subtilis group 1975 11.9.4 - + Not Tested
Bacillus subtilis group 1899 11.2.8 - + Not Tested Bacillus
subtilis group 1704 10.1.14 + - Not Tested Bacillus subtilis
group
[0092] The general success rate for selecting commercially viable
strains from a library of environmental spore-forming bacteria is
about 2%. With this genetic screening five of the 12 strains
selected were the same or better than the existing commercialized
strains i.e. a success rate of 42%.
[0093] Conclusions. Primers were developed to detect the nrsF gene
of the NRPD gene cluster and to the ycaO gene of the plantazolicin
operon, genes that were only present in the non-proprietary strain
2084. Using genetic screening twenty-nine strains were identified
from a library of over 2000 environmental spore-forming bacteria
for in vitro assays. Of twelve tested, five had similar or better
activity profiles to current proprietary strains, i.e. a success
rate of 42% compared to 2% without genetic preselection. Bacillus
strain 1999 was selected for commercialization.
Example 3
The Inhibitory Effect of Bacteriocins from Bacillus Strains 747,
1104, 1541, 1781 and 2018 on the Growth of Avian Pathogenic
Escherichia coli (APEC)
[0094] Introduction. Avian colibacillosis is a disease in chickens
caused by avian pathogenic Escherichia coli (APEC). Controlling or
reducing rates of colibacillosis in the commercial poultry industry
can have a significant economic impact (Georgopoulou et al., 2005).
Some strains of Bacillus have been shown to be effective in
preventing and controlling disease in poultry (La Ragione et al.,
2001; La Ragione and Woodward, 2003) This is likely in part due to
antimicrobial compounds commonly produced and secreted by many
Bacillus species such as bacteriocins (Tagg et al., 1976). Five
Bacillus strains selected for their antimicrobial properties
against Clostridium perfringens and APEC by the inventors.
Cell-free bacteriocin was collected from DFM strains 747, 1104,
1541, 1781, and 2018 and were used in a bioassay to determine their
inhibitory effect on the growth of a range of APEC strains isolated
from commercial broiler gastrointestinal tracts. This study was
conducted as an in vitro model in order to optimize DFM
formulations for use in a commercial broiler complex.
[0095] Materials and Methods. APEC Isolates: 28 APEC isolates,
harvested from broiler gastrointestinal tracts, were selected as
representatives from a commercial broiler complex. Genomic DNA was
extracted from each isolate using the following gDNA extraction
method: Add 20 .mu.L of lysozyme (100 mg/mL) to 500 .mu.L of
overnight growth in Tryptic Soy Broth (TSB; BD Difco) and incubate
at 37.degree. C. for 30 min, add 300 .mu.L of lysis buffer (6 M
Guanidine, 20% Triton-X 100, 10 mM Tris-HCl, pH 7.5) and incubate
at 25.degree. C. for 15 min, add 20 .mu.l of Protease K 800 U/ml
(NEB, Ipswich, Mass.) and incubate at 55.degree. for 30 min,
transfer 400 .mu.L of lysate to a Wizard.RTM. SV 96 Binding Plate
(Promega, Fitchburg, Wis.) and continue with manufacturer's
filtration instructions from Wizard.RTM. SV 96 Genomic DNA
Purification System starting from step 3.C.4 (4/15/revision)
(Promega, Fitchburg, Wis.).
[0096] APEC pathotype was determined using multiplex polymerase
chain reaction (mPCR). Each isolate was confirmed to contain at
least two of the five following APEC-associated virulence genes in
its genome: hlyF, ompT, iroN, iss, iutA. Each reaction mixture
contained 4 mM magnesium chloride (Invitrogen, Carlsbad, Calif.),
0.25 mM deoxynucleoside triphosphates (Invitrogen, Carlsbad,
Calif.), 0.25 .mu.M each primer (Eurofins, Brussels, Belgium), and
1 U Paltinum.RTM. Taq DNA Polymerase (Invitrogen, Carlsbad, Calif.)
and 5 .mu.L of template gDNA (Johnson et al., 2008). The reaction
was run on an Applied Biosystems Veriti.RTM. Thermal Cycler
(ThermoFisher Scientific, Milwaukee, Wis.) with the following
protocol: 94.degree. C. for 2 min; 25 cycles of 94.degree. C. for
30 s, 63.degree. C. for 30 s, 68.degree. C. for 3 min; and a final
cycle of 72.degree. C. for 10 min. The PCR product was then run
through capillary gel electrophoresis using a Fragment Analyzer.TM.
from Advance Analytical Technologies, Inc. and visualized on
PROsize 2.0 (Advanced Analytical Technologies, Inc., Ames,
Iowa).
[0097] The 28 APEC isolates used in this experiment were selected
from a pool of 136 total APEC isolates harvested from broiler
gastrointestinal tracts. The selected isolates were chosen as
cluster representatives based on a RAPD-PCR (RAPD primer 2
[5'-d{GTTTCGCTCC}-3']) similarity dendrogram in order to include
the broadest range of genetic variation between APEC isolates.
[0098] Bacteriocin: Each Bacillus strain (747, 1104, 1541, 1781,
and 2018) was grown from -80.degree. C. cell stock in 25 mL of TSB
in a 125 mL Erlenmeyer flask and incubated at 32.degree. C.,
shaking 150 rpm, for 24 h. 100 mL of TSB in a 500 mL Erlenmeyer
flask was inoculated with 1 mL of the 24-hour growth culture and
incubated 32.degree. C., shaking 150 rpm, for 36 h. After
incubation the growth culture was spun down at 14,000.times.g. The
supernatant was filter sterilized using 0.20 .mu.m, SFCA membrane
Nalgene.TM. Rapid-Flow.TM. vacuum filters and the filtrate was
frozen at -20.degree. C.
[0099] Bioassay: The select APEC isolates were grown up from frozen
stock by inoculating 10 mL tubes of TSB and incubating them at
37.degree. C. overnight. The resulting culture (100 .mu.L) was then
used to inoculate 10 mL tubes of TSB which were incubated at
37.degree. C. for 6 h to ensure growth-curve synchronicity. At the
start of the assay, TSB tubes were inoculated with 100 .mu.L of 6
hour growth and this inoculated media served as the indicator for
the assay. Aliquots of bacteriocin from each Bacillus strain were
thawed and gently mixed to guarantee homogeneity.
[0100] For every Bacillus strain, 70 .mu.L of bacteriocin was
dispensed into one well of a 48 well cell culture plate (Falcon,
Tewksbury, Mass.) for each of the 28 APEC isolates. For every APEC
isolate, 600 .mu.L of indicator media as described above was added
to the same wells containing Bacillus bacteriocin such that every
APEC isolate was paired with every Bacillus strain. Positive
control wells contained only 600 .mu.L of indicator, while negative
control wells contained 70 .mu.L of bacteriocin and 600 .mu.L of
fresh TSB to confirm bacteriocin sterility. The plate was incubated
at 37.degree. C., shaking at 150 rpm for 12-18 h.
[0101] After incubation, the OD of each well was read on a Biotek
Epoch Microplate Spectrophotometer at 600 nm wavelength. Results
were expressed as percent inhibition of APEC by Bacillus by using
the formula
( 1 - c x ) * 100 , ##EQU00001##
where C=blanked positive APEC control OD, and X=blanked treatment
OD.
[0102] Results. Percent inhibition of APEC isolates by bacteriocin
from Bacillus strains 747, 1104, 1541, 1781, and 2018 are shown
below in Table 3.
TABLE-US-00003 TABLE 3 Percent inhibition of APEC isolates by
Bacillus strains 747, 1104, 1541, 1781, and 2018. APEC 747 1104
1541 1781 2018 E50.1.2.2 86.0 55.8 27.1 93.1 93.3 E50.1.3.1 80.8
66.6 53.6 89.9 82.4 E50.1.3.2 68.3 47.3 31.6 62.0 53.9 E50.4.1.3
96.7 85.6 49.7 98.7 88.9 E50.6.2.2 68.6 60.6 37.4 83.7 47.3
E50.7.1.4 99.5 92.9 64.7 100.0 76.0 E50.7.2.1 100.0 99.3 55.6 100.0
97.9 E50.8.1.1 99.3 97.1 51.4 99.4 98.5 E50.8.1.3 100.0 92.8 32.4
100.1 99.5 E50.8.2.1 78.6 37.9 9.6 75.6 76.7 E50.9.2.3 78.5 59.4
40.0 78.8 66.3 E50.9.2.5 79.0 62.2 31.0 76.8 79.4 E50.9.3.3 85.6
66.3 19.3 73.0 56.3 E50.12.1.4 96.6 82.6 43.0 78.1 80.3 E50.12.1.5
95.6 74.5 21.7 80.5 83.6 E50.11.3.1 100.0 60.9 26.9 100.0 28.6
E50.14.1.3 73.2 55.5 23.3 74.0 61.4 E50.15.1.2 96.7 27.9 30.6 95.2
82.0 E50.16.2.1 75.5 25.0 0.0 59.1 38.9 E50.16.2.5 88.6 22.5 0.0
60.1 36.4 E50.17.2.1 99.6 96.8 74.4 97.7 95.2 E50.17.2.2 100.0 95.8
76.4 99.6 99.9 E50.17.2.3 100.0 94.8 61.5 100.0 99.8 E50.18.1.1
99.9 59.2 23.3 99.4 99.1 E50.18.3.1 100.0 99.9 50.7 100.0 100.0
E50.19.1.2 43.6 32.6 10.2 68.0 44.4 E50.20.1.4 75.0 62.4 40.2 81.1
58.9 E50.20.3.5 96.1 81.4 37.0 94.3 76.6 AVERAGE 87.9 67.7 36.5
86.4 75.0
[0103] All Bacillus strains showed inhibition of every APEC isolate
except for 1541 which showed no inhibition of two APEC isolates.
Bacillus strains 747 and 1781 exhibited the strongest overall
inhibitory effect with an average of 88.0% and 86.4% inhibition,
respectively, while 1541 presented the weakest inhibition with an
average of 35.2%.
[0104] Discussion. These results show clear evidence of an in vitro
inhibitory effect of avian pathogenic Escherichia coli by Bacillus
strains 747, 1104, 1541, 1781, and 2018. They also provide evidence
of a varied array of antimicrobial agents produced by each Bacillus
strain as indicated by the non-uniform pattern of inhibition across
the Bacillus strains. This suggests that implementation of multiple
Bacillus strains in combination could capture a greater breadth of
APEC genetic diversity and therefore be more effective in
preventing and controlling colibacillosis, while at the same time
avoiding selection of antimicrobial-resistant pathogens in a
commercial poultry setting.
Example 4
The Inhibitory Effect of Bacteriocins from Bacillus Strains 747,
1104, 1541, 1781 and 2018 on the Growth of Clostridium
perfringens
[0105] Introduction. Clostridium perfringens causes infection in
poultry known as Clostridium perfringens-associated necrotic
enteritis (NE). The most widespread toxinotype causing NE in
poultry is C. perfringens Type A, noted by its production of the C.
perfringens alpha toxin (Songer, 1996). C. perfringens-associated
NE can have a significant impact on commercial poultry operations
as it increases mortality and decreases weight gain of chickens and
turkeys, therefore controlling or reducing rates of NE in the
commercial poultry industry is highly desirable (Heier et al.,
2001; Lovland et al., 2003, 2004). Some strains of Bacillus have
been shown to be effective in preventing and controlling disease in
poultry (La Ragione et al., 2001). This is likely in part due to
antimicrobial compounds commonly produced and secreted by many
Bacillus species such as bacteriocins (Tagg et al., 1976). Five
Bacillus strains selected for their antimicrobial properties
against Clostridium perfringens and avian pathogenic E. coli (APEC)
were isolated by the inventors. Cell-free bacteriocin was collected
from DFM strains 747, 1104, 1541, 1781, and 2018 and were used in a
bioassay to determine their inhibitory effect on the growth of a
range of C. perfringens Type A strains isolated from commercial
broiler gastrointestinal tracts. This study was conducted as an in
vitro model in order to optimize DFM formulations for use in a
commercial broiler complex.
[0106] Materials and Methods. C. perfringens Type A Isolates: 18 C.
perfringens Type A isolates, harvested from broiler
gastrointestinal tracts, were selected as representatives from a
commercial broiler complex. Genomic DNA was extracted from each
isolate using the Roche Applied Science High Pure PCR Template Kit.
C. perfringens toxinotype was determined using polymerase chain
reaction (PCR) to amplify the alpha toxin gene. Each reaction
mixture contained 2.5 .mu.L 10.times.PCR buffer (Invitrogen,
Carlsbad, Calif.), 1.6 .mu.L magnesium chloride (Invitrogen,
Carlsbad, Calif.), 0.5 .mu.L deoxynucleoside triphosphates
(Invitrogen, Carlsbad, Calif.), 100 pmol primers (Eurofins,
Brussels, Belgium), and 1 U Paltinum.RTM. Taq DNA Polymerase
(Invitrogen, Carlsbad, Calif.) and 2 .mu.L of template gDNA, 7.8
.mu.L of ddH.sub.20 (Yoo et al., 1997). The reaction was run on an
Applied Biosystems Veriti.RTM. Thermal Cycler (ThermoFisher
Scientific, Milwaukee, Wis.) with the following protocol: 5 min at
94.degree. C., followed by 30 incubation cycles consisting of 1 min
at 55.degree. C., 1 min at 72.degree. C., and 1 min at 94.degree.
C. The PCR product was then run through capillary gel
electrophoresis using a Fragment Analyzer.TM. from Advance
Analytical Technologies, Inc. and visualized on PROsize 2.0
(Advanced Analytical Technologies, Inc., Ames, Iowa).
[0107] The 18 C. perfringens Type A isolates used in this
experiment were selected from a pool of 26 total C. perfringens
Type A isolates harvested from broiler gastrointestinal tracts. The
selected isolates were chosen as cluster representatives based on a
RAPD-PCR (RAPD primer 2 [5'-d{GTTTCGCTCC}-3']) similarity
dendrogram in order to include the broadest range of genetic
variation between C. perfringens Type A isolates.
[0108] Bacteriocin. Each Bacillus strain (747, 1104, 1541, 1781,
and 2018) was grown from -80.degree. C. cell stock in 25 mL of TSB
in a 125 mL Erlenmeyer flask and incubated at 32.degree. C.,
shaking 150 rpm, for 24 h. 100 mL of TSB in a 500 mL Erlenmeyer
flask was inoculated with 1 mL of the 24 hour growth culture and
incubated 32.degree. C., shaking 150 rpm, for 36 h. After
incubation the growth culture was spun down at 14,000.times.g. The
supernatant was filter sterilized using 0.20 .mu.m, SFCA membrane
Nalgene.TM. Rapid-Flow.TM. vacuum filters and the filtrate was
frozen at -20.degree. C.
[0109] Bioassay. The select C. perfringens Type A isolates were
grown up from frozen stock by inoculating 10 mL tubes of RCM broth
(Becton, Dickinson & Company) and incubating them at 37.degree.
C. overnight. The resulting culture (100 .mu.L) was then used to
inoculate 10 mL tubes of RCM which were incubated at 37.degree. C.
for 6 h to ensure growth-curve synchronicity. At the start of the
assay, RCM tubes were inoculated with 100 .mu.L of 6 hour growth
and this inoculated media served as the indicator for the assay.
Aliquots of bacteriocin from each Bacillus strain were thawed and
gently mixed to guarantee homogeneity.
[0110] For every Bacillus strain, 70 .mu.L of bacteriocin from each
Bacillus strain was dispensed into one well of a 48 well cell
culture plate (Falcon, Tewksbury, Mass.) for each of the 18 C.
perfringens Type A isolates. 600 .mu.L of indicator media (1%
inoculum in RCM) from each C. perfringens Type A isolate was added
to the same wells containing Bacillus bacteriocin such that every
C. perfringens Type A isolate was paired up with every Bacillus
strain. Positive control wells contained only 600 .mu.L of
indicator, while negative control wells contained 70 .mu.L of
bacteriocin and 600 .mu.L of un-inoculated RCM broth to confirm
bacteriocin sterility. The plate was incubated at 37.degree. C.,
anaerobically (Mitsubishi AnaeroPack System) for 12-18 h.
[0111] After incubation, the OD of each well was read on a Biotek
Epoch Microplate Spectrophotometer at 600 nm wavelength. Results
were expressed as percent inhibition of indicator isolate by
Bacillus by using the formula
( 1 - c x ) * 100 ##EQU00002##
where C=blanked positive indicator control OD, and X=blanked
treatment OD.
[0112] Results. Percent inhibition of C. perfringens Type A
isolates by bacteriocin from Bacillus strains 747, 1104, 1541,
1781, and 2018 are shown below in Table 4.
TABLE-US-00004 TABLE 4 Percent inhibition of Clostridium
perfringens Type A by Bacillus strains 747, 1104, 1541, 1781, and
2018 C. perfringens Type A 747 1104 1541 1781 2018 Isolate (%) (%)
(%) (%) (%) C50.18.2.1 98.7 98.5 98.6 98.9 98.2 C50.20.3.1 98.7
98.6 98.7 98.8 98.7 C50.20.3.2 98.7 98.5 98.7 98.8 98.6 C50.20.3.3
98.7 98.7 98.9 99.0 98.9 C50.20.3.4 98.8 98.7 98.7 99.1 98.8
C50.20.3.5 88.3 4.9 7.6 10.1 11.2 C50.22.2.1 98.3 98.3 98.3 98.4
98.6 C50.23.2.1 97.7 97.8 97.1 97.8 98.0 C50.23.2.2 95.6 95.0 95.0
95.3 95.5 C50.23.2.3 98.3 98.4 98.5 98.5 98.6 C50.23.2.4 95.2 94.4
94.8 94.9 95.0 C50.23.2.5 96.0 95.6 95.4 95.3 95.9 C50.24.2.1 95.1
94.2 94.4 94.1 94.2 C50.26.2.1 98.3 98.4 98.4 98.3 98.5 C50.26.2.2
95.5 94.7 95.3 95.0 95.4 C50.26.2.3 98.4 98.3 96.3 98.7 98.7
C50.26.2.5 98.2 98.5 94.7 98.4 98.4 C50.26.3.2 98.4 98.0 97.9 98.2
97.6 Average 97.1 92.2 92.1 92.6 92.7
[0113] All Bacillus strains showed significant inhibition (>85%)
of each C. perfringens Type A isolate with the exception of C.
perfringens Type A isolate C50.20.3.5 which was only significantly
inhibited by Bacillus strain 747. The highest average inhibition
was by Bacillus strain 747 with 97.1% and a range of 95.2% to
98.8%, while Bacillus strain 1541 exhibited the lowest average
inhibition with 92.1% and a range of 7.6% to 98.9%. Strain 1104
showed an average of 92.2% inhibition with a range of 4.9% to
98.7%, strain 1781 averaged 92.6% inhibition with a range of 10.1%
to 99.1%, and strain 2018 showed an average inhibition of 92.7%
with a range of 11.2% to 98.9%.
[0114] Discussion. These results show clear evidence of an in vitro
inhibitory effect of C. perfringens by Bacillus strains 747, 1104,
1541, 1781, and 2018. They also provide evidence of a varied array
of antimicrobial agents produced by each Bacillus strain as
indicated by the non-uniform pattern of C. perfringens inhibition
across the Bacillus strains. This suggests that implementation of
multiple Bacillus strains in combination could capture a greater
breadth of C. perfringens genetic diversity and therefore be more
effective in preventing and controlling C. perfringens-associated
diseases while at the same time avoiding selection of
antimicrobial-resistant pathogens in a commercial poultry
setting.
Example 5
The Effect of the Bacillus Direct Fed Microbial Product, According
to One Embodiment of the Present Invention, on the Gastrointestinal
Pathogen Load of Broilers
[0115] Introduction. The gastrointestinal-associated pathogens
Clostridium perfringens and avian pathogenic Escherichia coli
(APEC) can have significant negative ramifications on the
productivity of commercial broiler operations (Georgopoulou et al.,
2005). C. perfringens strains that produce alpha toxin are
categorized as the C. perfringens Type A toxinotype and cause
necrotic enteritis in poultry which increases mortality and reduces
weight gain (Immerseel et al., 2004). APEC is a causative agent for
colibacillosis in birds in the form of airsacculitis, cellulitis,
pericarditis, or perihepatitis (Barnes H J et al., 2008).
Colibacillosis infections are of considerable concern for the
poultry industry as they are the responsible for high rates of bird
death and are the most reported reason for processing rejection
(Yogaratnam, 1995). Controlling or reducing rates of NE and
colibacillosis in the commercial broiler industry can increase
efficiency and productivity which may bare substantial economic
impacts for poultry growers. Some strains of Bacillus have been
shown to be effective in preventing and controlling disease in
poultry (La Ragione and Woodward, 2003; La Ragione et al., 2001).
This is likely in part due to antimicrobial compounds commonly
produced and secreted by many Bacillus species such as bacteriocins
(Tagg et al., 1976). Five Bacillus strains selected for their
antimicrobial properties against Clostridium perfringens and avian
pathogenic E. coli (APEC) were isolated by the inventors. These
five Bacillus strains were commercialized for use as DFM in poultry
feed. By surveying pathogens in the gastrointestinal tracts of
broilers from a commercial complex before and after treatment with
the Bacillus DFM product, according to one embodiment of the
present invention, a significant reduction in pathogen load for
both C. perfringens and APEC was detected.
[0116] Design. Gastrointestinal tracts (GIT) from broiler chickens
were sampled from a variety of houses within a commercial broiler
complex before and after implementation of the Bacillus DFM
product, according to one embodiment of the present invention.
Sampling 1 consisted of 22 GITs (average age 22 d) before product,
while sampling 2 had 24 GITs (average age 25 d) on treated
feed.
[0117] Materials and Methods. Direct fed microbial: Treated birds
were given feed supplemented with a formulation of Bacillus strains
1104 (40%), 1541 (20%) and 1781 (40%) at a final concentration of
1.5.times.10.sup.5 CFU/g in finished feed.
[0118] Processing of Gastrointestinal Tracts: Selected broilers
were sacrificed and the gastrointestinal tracts from the duodenal
loop to the cloaca were removed and transported in sterile
Whirl-pak.RTM. bags on ice. Upon arrival, 10 cm sections of the
duodenum, jejunum, and ilium were rinsed with .about.5 mL sterile
0.1% peptone broth, cut longitudinally, and combined in a sterile,
filtered whirl-pak bag. 99 mL of sterile 0.1% peptone was added to
the bag then the sections were masticated at 300 rpm, for 1 min.
Serial dilutions were made and pour plated in duplicate with both
CHROMagar.TM. ECC to enumerate E. coli, and perfringens TSC agar
base (Oxoid.TM.) with D-cycloserine (Sigma, 400 mg/L) for
Clostridium enumeration.
[0119] APEC Screening: Typical E. coli colonies on CHROMagar.TM.
appear blue. After 12-24 h of incubation at 37.degree. C., all blue
colonies were counted and recorded as presumptive APEC CFU/g
counts. Five isolated blue colonies from each bird were picked and
enriched in TSB (Becton, Dickenson & Company, Franklin Lakes,
N.J.) if possible. Genomic DNA was extracted from each isolate
using the following gDNA extraction method: Add 20 .mu.L of
lysozyme (100 mg/mL) to 500 .mu.L of overnight growth in TSB and
incubate at 37.degree. C. for 30 min, add 300 .mu.L of lysis buffer
(6 M Guanidine, 20% Triton-X 100, 10 mM Tris-HCl, pH 7.5) and
incubate at 25.degree. C. for 15 min, add 20 .mu.l of Protease K
800 U/ml (NEB, Ipswich, Mass.) and incubate at 55.degree. for 30
min, transfer 400 .mu.L of lysate to a Wizard.RTM. SV 96 Binding
Plate (Promega, Fitchburg, Wis.) and continue with manufacturer's
filtration instructions from Wizard.RTM. SV 96 Genomic DNA
Purification System starting from step 3.C.4 (4/15/revision)
(Promega, Fitchburg, Wis.).
[0120] APEC pathotype was determined using multiplex polymerase
chain reaction (mPCR). In order to be considered APEC, an E. coli
isolate had to contain at least two of the five following
APEC-associated virulence genes in its genome: hlyF, ompT, iroN,
iss, iutA. Each reaction mixture contained 4 mM magnesium chloride
(Invitrogen, Carlsbad, Calif.), 0.25 mM deoxynucleoside
triphosphates (Invitrogen, Carlsbad, Calif.), 0.25 .mu.M each
primer (Eurofins, Brussels, Belgium), and 1 U Paltinum.RTM. Taq DNA
Polymerase (Invitrogen, Carlsbad, Calif.) and 5 .mu.L of template
gDNA (Johnson et al., 2008). The reaction was run on an Applied
Biosystems Veriti.RTM. Thermal Cycler (ThermoFisher Scientific,
Milwaukee, Wis.) with the following protocol: 94.degree. C. for 2
min; 25 cycles of 94.degree. C. for 30 s, 63.degree. C. for 30 s,
68.degree. C. for 3 min; and an extension cycle of 72.degree. C.
for 10 min. The mPCR product was then run through capillary gel
electrophoresis using a Fragment Analyzer.TM. from Advance
Analytical Technologies, Inc. and visualized on PROsize 2.0
(Advanced Analytical Technologies, Inc., Ames, Iowa).
[0121] C. perfringens Type A Screening: Presumptive C. perfringens
isolates appear black on perfringens TSC agar base. All black
colonies were counted and recorded as presumptive C. perfringens
CFU/g counts. Five isolated black colonies from each bird were
picked and grown in RCM broth (Oxoid.TM.) if possible. Genomic DNA
was extracted from each isolate using the Roche Applied Science
High Pure PCR Template Kit.
[0122] C. perfringens toxinotype was determined using polymerase
chain reaction (PCR) to amplify the alpha toxin gene. In order for
an isolate to be considered Clostridium perfringens Type A it had
to contain the alpha toxin gene, otherwise it was categorized as a
non-perfringens Clostridium. Each reaction mixture contained 2.5
.mu.L 10.times.PCR buffer (Invitrogen, Carlsbad, Calif.), 1.6 .mu.L
magnesium chloride (Invitrogen, Carlsbad, Calif.), 0.5 .mu.L
deoxynucleoside triphosphates (Invitrogen, Carlsbad, Calif.), 100
pmol primers (Eurofins, Brussels, Belgium), and 1 U Paltinum.RTM.
Taq DNA Polymerase (Invitrogen, Carlsbad, Calif.), 2 .mu.L of
template gDNA, and 7.8 .mu.L of ddH.sub.20 (Yoo et al., 1997). The
reaction was run on an Applied Biosystems Veriti.RTM. Thermal
Cycler (ThermoFisher Scientific, Milwaukee, Wis.) with the
following protocol: 5 min at 94.degree. C., followed by 30
incubation cycles consisting of 1 min at 55.degree. C., 1 min at
72.degree. C., and 1 min at 94.degree. C. The PCR product was then
run through capillary gel electrophoresis using a Fragment
Analyzer.TM. from Advance Analytical Technologies, Inc. and
visualized on PROsize 2.0 (Advance Analytical Technologies,
Inc).
[0123] Counts and Statistics: Pathotype levels for each bird were
determined by multiplying the weight-adjusted presumptive CFU/g
counts by the percent of presumptive isolates from each bird that
were revealed to be pathogenic (C. perfringens or APEC). Birds that
did not produce any detectable colonies on agar plates or birds
that did not produce any confirmed pathogens through screening were
assigned a value of <500 CFU/g for APEC and <50 CFU/g for
Clostridium perfringens (for calculations this value was entered as
500 CFU/g and 50 CFU/g, respectively).
[0124] Statistical analysis for the comparison of untreated vs
treated birds was run using an unpaired two-tailed t-test.
Significant difference threshold was set at P<0.05.
[0125] Results. The pathogen counts represented in CFU/g of tissue
are shown in FIGS. 1 and 2. Birds on treated feed exhibited a
significant APEC reduction with an average APEC level of
2.1.times.10.sup.4 CFU/g compared to birds on untreated feed which
yielded an average APEC level of 2.1.times.10.sup.5 CFU/g. APEC
levels of untreated birds ranged from 5.0.times.10.sup.3 to
2.2.times.10.sup.6 CFU/g while treated birds ranged from
5.0.times.10.sup.3 to 6.6.times.10.sup.5 CFU/g.
[0126] Clostridium perfringens levels of birds fed untreated feed
showed an average level of 1.7.times.10.sup.2 CFU/g. Levels from
treated birds, by comparison, were below detectable limits for all
birds, therefore average level of C. perfringens for treated birds
was calculated as 50 CFU/g.
[0127] Discussion. These data demonstrate significant reduction of
APEC and Clostridium perfringens levels in broilers GITs fed the
Bacillus DFM product, according to one embodiment of the present
invention. Reduction of these pathogens can diminish cases of
disease in broilers such as avian colibacillosis and necrotic
enteritis, diseases which present significant financial liability
to the poultry industry. Our research shows that including the
Bacillus DFM product, according to one embodiment of the present
invention, in feed is effective in reducing APEC and C. perfringens
prevalence in broilers, therefore decreasing the disease-burden in
commercial broiler operations.
Example 6
The Effect of the Bacillus DFM Product, According to One Embodiment
of the Present Invention, on the Gastrointestinal Pathogen Load of
Turkeys
[0128] Introduction. The gastrointestinal-associated pathogen
Clostridium perfringens can have significant negative ramifications
on the productivity of commercial poultry operations. C.
perfringens strains that produce alpha toxin are categorized as the
C. perfringens Type A toxinotype and cause necrotic enteritis in
poultry which increases mortality and reduces weight gain (Lovland
et al., 2004) Thus, controlling or reducing rates of NE in the
commercial turkey industry can increase efficiency and productivity
which may bare substantial financial impacts to turkey growers.
Some strains of Bacillus have been shown to be effective in
preventing and controlling disease in poultry (La Ragione et al.,
2001; La Ragione and Woodward, 2003). This is likely in part due to
antimicrobial compounds commonly produced and secreted by many
Bacillus species such as bacteriocins (Tagg et al., 1976). Five
Bacillus strains selected for their antimicrobial properties
against Clostridium perfringens and avian pathogenic E. coli (APEC)
were isolated by the inventors. These five Bacillus strains (747,
1104, 1541, 1781, and 2018) were commercialized for use as DFM in
poultry feed. By surveying pathogens in the gastrointestinal tracts
of turkeys from a commercial complex before and after treatment
with the Bacillus DFM product, according to one embodiment of the
present invention, a significant reduction in C. perfringens
pathogen load was detected.
[0129] Design. Turkey gastrointestinal tracts (GIT) were sampled
from a variety of houses within a commercial turkey complex before
and after implementation of the Bacillus DFM product, according to
one embodiment of the present invention. Sampling 1 consisted of 18
GITs (average age 60 d) before product, while sampling 2 had 24
GITs (average age 34 d) on treated feed. Birds taken on sampling 2
were treated with a formulation of the Bacillus DFM product,
according to one embodiment of the present invention.
[0130] Materials and Methods. Direct fed microbial: Treated birds
were given feed supplemented with a formulation of the Bacillus
strain 1104 (50%) identified herein and commercially available
strain Bs2084 (Microbial Discovery Group, Franlkin, Wis.) at a
final concentration of 1.5.times.10.sup.5 CFU/g of finished
feed.
[0131] Processing of Gastrointestinal Tracts: Selected turkeys were
sacrificed and the gastrointestinal tracts from the duodenal loop
to the cloaca were removed and delivered in sterile Whirl-pak.RTM.
bags on ice. Upon arrival, 10 cm sections of the duodenum, jejunum,
and ilium were rinsed with .about.5 mL sterile 0.1% peptone broth,
cut longitudinally, and combined in a sterile, filtered whirl-pak
bag. 99 mL of sterile 0.1% peptone was added to the bag then the
sections were masticatedat 300 rmp, for 1 min. Samples were heat
shocked at 60.degree. C. for 30 min then serial dilutions were made
and pour-plated in duplicate with perfringens TSC agar base
(Oxoid.TM.) with D-cycloserine (Sigma, 400 mg/L) for Clostridium
enumeration.
[0132] C. perfringens Type A Screening: Presumptive C. perfringens
isolates appear black on perfringens TSC agar base. All black
colonies were counted and recorded as presumptive C. perfringens
CFU/g counts. Five isolated black colonies from each bird were
picked and grown in RCM broth (Oxoid.TM.) if possible. Genomic DNA
was extracted from each isolate using the Roche Applied Science
High Pure PCR Template Kit.
[0133] C. perfringens toxinotype was determined using polymerase
chain reaction (PCR) to amplify the C. perfringens alpha toxin
gene. In order for an isolate to be considered Clostridium
perfringens Type A it had to contain the alpha toxin gene,
otherwise it was categorized as a non-perfringens Clostridium. Each
reaction mixture contained 2.5 .mu.L 10.times.PCR buffer
(Invitrogen, Carlsbad, Calif.), 1.6 .mu.L magnesium chloride
(Invitrogen, Carlsbad, Calif.), 0.5 .mu.L deoxynucleoside
triphosphates (Invitrogen, Carlsbad, Calif.), 100 pmol primers
(Eurofins, Brussels, Belgium), and 1 U Paltinum.RTM. Taq DNA
Polymerase (Invitrogen, Carlsbad, Calif.) and 2 .mu.L of template
gDNA, 7.8 .mu.L of ddH.sub.20 (Yoo et al., 1997). The reaction was
run on an Applied Biosystems Veriti.RTM. Thermal Cycler
(ThermoFisher Scientific, Milwaukee, Wis.) with the following
protocol: 5 min at 94.degree. C., followed by 30 incubation cycles
consisting of 1 min at 55.degree. C., 1 min at 72.degree. C., and 1
min at 94.degree. C. before a final extension at 72.degree. C. for
7 minutes. The PCR product was then run through capillary gel
electrophoresis using a Fragment Analyzer.TM. from Advance
Analytical Technologies, Inc. and visualized on PROsize 2.0
(Advanced Analytical Technologies, Inc., Ames, Iowa).
[0134] Counts and Statistics: Pathotype levels for each bird were
determined by multiplying the weight-adjusted presumptive C.
perfringens Type A CFU/g counts by the percent of presumptive
isolates from each bird that were revealed to possess the C.
perfringens alpha toxin. Birds that did not produce any detectable
colonies on agar plates or birds that did not produce any confirmed
alpha-toxin-producing isolates through screening were assigned a
value of <50 CFU/g (for calculations this value was entered as
50 CFU/g).
[0135] Statistical analysis for the comparison of untreated vs
treated birds was run using an unpaired two-tailed t-test.
Significant difference threshold was set at P<0.05.
[0136] Results. Clostridium perfringens counts represented in CFU/g
of tissue are shown in FIG. 3. Clostridium perfringens levels of
birds fed untreated feed showed an average level of
1.7.times.10.sup.2 CFU/g with a range of 50 to 5.5.times.10.sup.4
CFU/g. Levels from treated birds, by comparison, were below
detectable limits for all birds, therefore average level of C.
perfringens for treated birds was calculated as 50 CFU/g.
[0137] Discussion. These data demonstrate a significant reduction
of Clostridium perfringens levels in turkeys GITs fed the Bacillus
DFM product, according to one embodiment of the present invention.
Reduction of this pathogen can diminish cases of necrotic enteritis
in turkeys, a disease which presents a significant financial
liability for the poultry industry. Our research shows that
including the Bacillus DFM product, according to one embodiment of
the present invention, in feed is effective in reducing C.
perfringens prevalence in turkeys, therefore decreasing the
disease-burden in commercial broiler operations.
Example 7
Comparison of Poultry Bacillus Genomes
[0138] Introduction. Many bacteria produce compounds that can
inhibit other bacteria, commonly known as bacteriocins. The
function of bacteriocins is to allow the producer cells to compete
with other microbes in their natural environment. They generally
increase membrane permeability by forming pores in membranes of
target cells or inhibit cell wall synthesis thereby preventing
growth of susceptible microbes. Bacillus species produce multiple
compounds with inhibitory activity against other microbes with many
strains containing more than ten operons producing antifungal and
antibacterial compounds. These compounds may have other functions
within the cell. For example, surfactin is involved in
intercellular signaling and biofilm formation (Zeriouh et al.,
2014) and bacillibactin, is an iron-scavenger for Bacillus species,
which then deprives other organisms of essential iron and inhibits
their growth (Li et al., 2014).
[0139] The predominant bacteriocins produced by bacilli are a
variety of functionally and structurally diverse peptides. They are
often hydrophobic and cyclic with unusual amino acids and resistant
to peptidases and proteases. They may be synthesized ribosomally or
nonribosomally by multi-enzyme complexes, often followed by
post-translational modifications.
[0140] A major group of ribosomally synthesized antimicrobial
peptides are the lantibiotics, which contain the non-protein amino
acid lanthionine, formed post-translationally. Type A lantibiotics
have a linear secondary structure while Type B are more
globular.
[0141] Bacillus strains often produce nonribosomally synthesized
lipopeptides, fatty acids attached to small cyclic peptides. These
nonribosomally synthesized peptides are structurally diverse (Luo
et al., 2015a), as they are assembled from a heterogeneous group of
precursors, but their synthesis by a multicarrier thiotemplate
mechanism is conserved (Luo et al., 2015b). Nonribosomal peptide
synthetases (NRPS) require posttranslational modification to be
functionally active.
[0142] Other non-peptide antimicrobials are also produced by
bacilli such as polyketides and siderophores. Polyketides are
secondary metabolites which are also synthesized on multienzymes
similar to NRPS and they also undergo posttranslational
modification. Hybrid synthetases containing peptide, fatty acid and
polyketide synthetase domains are also being identified in bacilli
and some of these compounds were shown to have functional
antimicrobial activity.
[0143] Most of the information available on the secondary compounds
produced by bacilli and their antimicrobial activity (Chowdhury et
al., 2015; Koumoutsi et al., 2004) is based on studies on
plant-growth promoting rhizobacteria (PGPR) that are applied as
spore formulations to improve crop production by promoting growth
and inhibiting plant pathogens (Wu et al., 2015). A better
understanding of how an organism lives and competes in its
environment can be obtained by sequencing their full genome. Since
1995, when the first complete genome sequence of the bacteria
Haemophilus influenzae Rd KW20 was published (Fleischmann et al.,
1995), sequencing of genomes has increased exponentially and
powerful databases and bioinformatics programs have been developed
in order to predict the functions of newly sequenced organisms.
Gene function is predicted based on the genetic organization of
surrounding genes, conserved protein domains within genes and
alignment with genes of established function. Predicted gene
functions should then be confirmed by further molecular and
biochemical experiments. A number of genome sequences of PGPR are
available (Chen et al., 2007; Jeong et al., 2015) and the core
genome and conserved antimicrobial loci have been identified (Fan
et al., 2015). Comparative analysis of the draft genomes of the
Bacillus strains identified herein (747, 1104, 1541, 1781, 1999,
and 2018) to genomes available in the databases allowed the
inventors to predict the types of antimicrobial compounds produced
by the strains and determine differences between strains.
[0144] Methods. Draft genomes were obtained for six Bacillus
strains identified herein (747, 1104, 1541, 1781, 1999, and 2018)
and six previously known strains of bacilli (Baltzley et al., 2010)
by assembling paired-end Illumina reads of genomic DNA. Shotgun
genomic libraries were prepared with the library construction kit
from Kapa Biosystems with an average gDNA fragment size of 435 bp
(320-600 bp). The library pool was sequenced on one MiSeq flowcell
for 261 cycles generating 261 nt reads using a MiSeq sequencing kit
v2 (Illumina, San Diego, Calif.). Paired reads were merged to
generate longer single reads with PEAR 0.9.6 (Zhang et al., 2014).
High quality reads that passed data preprocessing steps were
assembled with SPAdes 3.5.0 assembler (Nurk et al., 2013). The
assembled scaffolds ranged from 18 to 51 contigs and were annotated
using the prokka 1.11 annotation pipeline (Seemann, 2014). The
draft genomes were between 3.91 and 4.15 Mb in size with 3894 to
4212 predicted genes as shown below is Table 5.
TABLE-US-00005 TABLE 5 General characteristics of the draft genomes
obtained for the Bacillus strains Bacillus Strains according to one
embodiment of the present invention Prior Known Bacillus Strains
747 1104 1541 1781 1999 2018 15A-P4 2084 22C-P1 3A-P4 LSSA01 Bs27
Size (Mb) 4.04 4.11 3.91 4.05 3.92 3.96 4.15 3.92 4.09 3.93 4.05
3.92 Number of Contigs 38 42 32 36 32 41 38 46 51 39 51 18 % GC
46.2 46.1 46.4 46.2 46.5 46.5 45.9 46.5 46.3 46.5 46.4 45.8
Annotated Genes 4065 4180 3894 4066 3953 3894 4212 3933 4141 3927
3949 4155 Protein-coding 3920 4028 3748 3920 3775 3748 4052 3776
4003 3780 3778 3995 genes Genes with 2986 3068 2938 2987 2971 2938
3044 2971 3064 2982 2968 2981 predicted functions Hypothetical
genes 934 960 810 933 804 810 1008 805 939 798 810 1014 Genes with
signal 275 274 263 274 255 263 277 256 275 267 255 285 peptides
[0145] The contigs of each draft genome were aligned and ordered
with Mauve genome alignment software (Darling et al., 2010) against
a fully sequenced genome and then concatenated with a 100 Ns
demarcating the contig boundaries. The concatenated draft genomes
were compared using various tools in Geneious 8.1.7 (Kearse et al.,
2012), EDGAR 2.0 (Blom et al., 2016) and PATRIC (Wattam et al.,
2014).
[0146] Results. Referring now to FIG. 4, a phylogenetic tree based
upon similarities and differences in the draft genomes was created
by MAUVE alignment, which indicates the relatedness of each of the
strains to the others, is provided. This tree indicates that the
strains 747 and 1781 are closely related, as are 1999, 2084 and
LSSA01.
[0147] EDGAR Venn diagrams were created to identify unique genes in
five of the Bacillus identified herein compared to the previously
known strains. As only five strains can be compared in a Venn
diagram previously known strain Bs27 was not included as it is the
most different to any of the strains and previously known strain
LSSA01 was not included as it is genetically similar to 2084. The
majority of the genes identified as being unique to the strains
were annotated as hypothetical i.e. there is no predicted function
associated with the genes as shown below in Table 6. Many of these
genes were clustered together in the genome indicating that they
are likely part of a metabolic pathway.
TABLE-US-00006 TABLE 6 Genes that are present only in the Bacillus
strains, according to the present, compared to the prior strains
3A-P4, 15A-P4, 2084 and 22C-P1. The majority of the unique genes
are annotated as hypothetical, some of which are associated in
clusters that likely represent unique metabolic pathways. Strain
747 1104 1541 1781 2018 Unique genes 188 92 113 188 40 Hypothetical
genes 135 63 71 135 22 Unique gene clusters 28 16 18 28 8
[0148] A detailed analysis of all twelve genomes indicated that
gene clusters for a number of antimicrobial secondary metabolites
were present in one or more strains, but absent in others as shown
below in Table 7.
TABLE-US-00007 TABLE 7 Operons associated with antimicrobial
production that differ among Bacillus strains. Size
Metabolite/Function No. of genes (kb) 747 1104 1541 1781 1999 2018
2084 LSSA01 3A-P4 22C-P1 15A-P4 Bs27 Presumptive Lantibiotic 1 13
15.6 + + - + - + - - + - - - Presumptive Lantibiotic 2 7 9.5 - - +
- - - - - - - - - Bacillomycin D 4 30.2 - - + - + - + + - - + +
Iturin A 4 30.2 + + - + - + - - + + - - Plispastatin (formerly 5
37.6 + + + + + + + + + + + - fengycin) Presumptive Gramicidin 8
31.5 + - - + - - - - - - - - Presumptive non- 6 17.5 - - - - + - +
+ - - - - ribosomal antimicrobial Plantazolicin 11 9.96 - - - - + -
+ + - - - - Bacitracin Export 2 2 2.7 - + - - - - - - + + - -
[0149] A related gene cluster containing genes annotated as
lanthionine synthetases was present in strains 747, 1781, 1104,
2018 and the previously known strain 3A-P4. A seven gene cluster
containing lanthionine synthetases was unique to 1541. A polyketide
synthesizing region, presumptively identified as gramicidin, was
present only in 747 and 1781.
[0150] All the strains were predicted to produce an Iturin A-like
non-ribosomally synthesized lipopeptide, but for some strains the
genes were more similar to Bacillomycin D producers and for the
others Iturin A producers. Genes predicted to code for plipastatin
(formerly known as fengycin) were absent in strain Bs27, but
present in the others. Genes for a presumptive non-ribosomally
synthesized antimicrobial and plantazolicin, a post-translationally
modified peptide, were detected in the 1999 and the previously
known strains 2084 and LSSA01.
[0151] Genes annotated as bacitractin transporters, which are
predicted to export one or more of the antimicrobials produced,
were present in all strains, however a second region was present in
1104, 3A-P4 and 22C-P1.
[0152] There were some antimicrobial-associated gene clusters that
were present in all the strains compared. These regions were not
identical but had a nucleotide similarity greater than 97% across
the region. These were two non-ribosomally synthesized
lipopeptides, surfactin and bacilysin, and three polyketides,
bacillaene, macrolactin and difficidin. The post-translationally
modified peptide, amylocyclin, as well as the siderophore,
bacillibactin, were also present in all strains.
[0153] Variation analysis to annotate single nucleotide
polymorphism (SNP), multiple nucleotides polymorphism (MNP) and
insertion or the deletion of bases (indel) sequence variation
between closely related strains, was run in PATRIC to identify
differences between 1999 and the previously known strains 2084 and
LSSA01, as well as the two Bacillus strains 747 and 1781, according
to one embodiment of the present invention. In total 60
high-quality variants were found between the Bacillus strain 1999,
according to the present invention, and the two commercially
available strains 2084 and LSSA01 as shown below in Table 8.
TABLE-US-00008 TABLE 8 Nucleotide (nt) variations between Bacillus
strain 1999, according to the present invention and prior strains
2084 and LSSA01 showing the amino acid (aa) substitutions and the
functions of the genes affected by the genetic differences between
the three strains. Genome Type of Position 1999 2084 LSSA01 Amino
Acid 1999 Var 1999 Var in 1999 nt nt nt Substitution nt nt aa aa
Function 233023 T C T Synonymous gta gtG V V Lincomycin-resistance
313465 CA TAAC TAAC Synonymous tacaat taTAAC YN YN Surfactin
production and AT 313534 C T T Synonymous gac gaT D D Surfactin
production and competence 314473 A C A Synonymous gca gcC A A
Surfactin production and competence 314488 G C G Synonymous cc g
ccC P P Surfactin production and competence 314725 TG CGA CGA
Nonsynonymous attgaag atCGAAA IE IE Surfactin production and AA AA
AA cg cg A T competence G 627977 A G G Intergenic region 710497 T A
A Nonsynonymous cag cTg Q L Glutamine synthetase family protein
710690 T T C Nonsynonymous agc Ggc S G Glutamine synthetase family
protein 710721 C G C Nonsynonymous cag caC Q H Glutamine synthetase
family protein 710727 CA TAAC TAAC Nonsynonymous ggcctgt gACCAG GL
DQ Glutamine synthetase AC TGGT TGGT tg TTA L L family protein AG
GC 710747 AC AGC AGC Insertion ccgttt ccGCTtt PF PL Glutamine
synthetase family protein 710770 G T T Nonsynonym gct gAt A D
Glutamine synthetase ous family protein 714445 A G G Synonymous att
atC I I Peptidase S8 and S53, subtilisin, kexin, sedolisin 714451 C
G G Synonymous cc g ccC P P Peptidase S8 and S53, subtilisin,
kexin, sedolisin 714490 A C C Synonymous gct gcG A A Peptidase S8
and S53, subtilisin, kexin, sedolisin 714547 A T T Synonymous gtt
gtA V V Peptidase S8 and S53, subtilisin, kexin, sedolisin 1502309
A G A Synonymous ggt ggC G G 2-oxoglutarate dehydrogenase E1
component (EC 1.2.4.2) 2243964 C G C Nonsynonymous ggc gCc G A
Stage V sporulation protein AD (SpoVAD) 2264062 G A G Nonsynonymous
atg atA M I YqkD 2306297 A C C Nonsynonymous ttc Gtc F V Polyketide
synthase 2306325 T G G Synonymous aca acC T T Polyketide synthase
2306658 A A G Synonymous att atC I I Polyketide synthase 2306765 C
T T Nonsynonymous ggc Agc G S Polyketide synthase 2306778 G A A
Synonymous tac taT Y Y Polyketide synthase 2306791 G G T
Nonsynonymous gcg gAg A E Polyketide synthase 2306812 C C T
Nonsynonymous ggt gAt G D Polyketide synthase 2306817 C C T
Synonymous ccg ccA P P Polyketide synthase 2306827 C C T
Nonsynonymous cgg cAg R Q Polyketide synthase 2306882 A A G
Nonsynonymous tgt Cgt C R Polyketide synthase 2307018 A A G
Synonymous gct gcC A A Polyketide synthase 2307048 G G A Synonymous
tcc tcT S S Polyketide synthase 2307210 C A C Synonymous tcg tcT S
S Polyketide synthase 2307077 T T A Nonsynonymous act Tct T S
Polyketide synthase 2307281 TGC CGCA CGC Nonsynonymous cgctgca
cgTTGCG RC RC Polyketide synthase AG A AA gc gc S G 2307296 G C C
Nonsynonymous ctt Gtt L V Polyketide synthase 2307312 TAC AAC AAC
Synonymous ccggta ccTGTT PV PV Polyketide synthase C A A 2307324 C
T T Synonymous gag gaA E E Polyketide synthase 2307362 T C C
Nonsynonymous atc Gtc I V Polyketide synthase 2307525 G A A
Synonymous gcc gcT A A Polyketide synthase 2307545 C A A
Nonsynonymous gcc Tcc A S Polyketide synthase 2466542 G A G
Nonsynonymous ccg cTg P L Nonribosomal lipopeptide synthetase
2475614 G G A Synonymous gac gaT D D Polyketide synthase 2475665 T
T C Synonymous caa caG Q Q Polyketide synthase 2776890 T T G
Synonymous aca acC T T Hypothetical protein formerly called
flagellar hook-length control protein FliK 2776908 A T T Synonymous
act acA T T Hypothetical protein formerly called flagellar
hook-length control protein FliK 2856746 G A A Synonymous ggc ggT G
G Nonribosomal lipopeptide synthetase 2857890 G A A Nonsynonymous
act aTt T I Nonribosomal lipopeptide synthetase 2858025 G A A
Nonsynonymous gca gTa A V Nonribosomal lipopeptide synthetase
2858056 C T T Nonsynonymous gat Aat D N Nonribosomal lipopeptide
synthetase 2858068 G C C Nonsynonymous ctg Gtg L V Nonribosomal
lipopeptide synthetase 2858081 C T T Synonymous agg agA R R
Nonribosomal lipopeptide synthetase 2858117 G A A Synonymous gcc
gcT A A Nonribosomal lipopeptide synthetase 2858246 G A A
Synonymous gtc gtT V V Nonribosomal lipopeptide synthetase 2858459
C T T Synonymous gag gaA E E Nonribosomal lipopeptide synthetase
3290611 C C T Nonsynonymous cgt cAt R H FIG000506: Predicted P-
loop-containing kinase 3387134 G A G Nonsynonymous ccg cTg P L
Glycosyltransferase 3913269 TG TAG TG Insertion Intergenic region
3913453 G T T Synonymous nng nnT X X Hypothetical protein 3925358 C
T T Nonsynonymous gcc gTc A V Nonribosomal lipopeptide
synthetase
[0154] There were 20 high-quality variants between 747 and 1781 as
shown below in Table 9.
TABLE-US-00009 TABLE 9 Nucleotide (nt) variations between the
Bacillus 747 and 1781 strains according to the present invention,
showing the amino acid (aa) substitutions and the functions of the
genes affected by the genetic differences between the two strains.
Position 747 1781 Amino Acid 747 1781 747 1781 in 747 nt nt
Substitution nt nt2 aa aa Function 129 C T Intergenic region 484 C
G Nonsynonymous caa Gaa Q E 3-oxoacyl-[cyl-carrier- protein]
synthase, KASII (EC 2.3.1.179) 17244 C T Nonsynonymous gcg gTg A V
Chromosome (plasmid) partitioning protein ParB-2 46296 G A
Nonsynonymous ggt gAt G D Nonribosomal lipopeptide synthetase
196876 T C Synonymous ggt ggC G G Collagen adhesion protein 197027
A G Nonsynonymous aag Gag K E Collagen adhesion protein 197116 GT
CC Nonsynonymous gtg gtC V V Collagen adhesion protein T G ttg CGg
L R 197164 T C Synonymous gtt gtC V V Collagen adhesion protein
197179 TA CC Nonsynonymous gat gaC D D Collagen adhesion protein
agt Cgt S R 197193 TA CG Nonsynonymous ata aCG I T Collagen
adhesion protein 197221 G A Synonymous aag aaA K K Collagen
adhesion protein 197251 CC AC Synonymous gcc gcA A A Collagen
adhesion protein CG CC ccg CCC P P 197266 C A Synonymous gcc gcA A
A Collagen adhesion protein 197272 C T Synonymous atc atT I I
Collagen adhesion protein 209653 T C Synonymous ttt ttC F F
Surfactin production and competence 209707 A G Synonymous gaa gaG E
E Surfactin production and competence 285301 G A Intergenic region
534678 T G Nonsynonymous ctg cGg L R Sensor protein DegS 732601 C G
Nonsynonymous gaa Caa E Q Phage-like element PBSX protein xkdE
816742 C A Intergenic region
[0155] Conclusions. An in depth comparison of the genomes of the
Bacillus strains, according to one embodiment of the present
invention, to previously commercially available strains indicates
that there are multiple genetic differences between all the
strains. Functionally and genetically, the Bacillus strains,
according to one embodiment of the present invention, are different
to the previously commercially available strains.
Example 8
The Inhibition of APEC by a Direct Fed Bacillus Strain, According
to One Embodiment of the Present Invention, is Maintained Over
Time
[0156] Introduction. Avian colibacillosis is a disease in chickens
caused by avian pathogenic Escherichia coli (APEC). Controlling or
reducing rates of colibacillosis in the commercial poultry industry
can have a significant economic impact (Georgopoulou et al., 2005).
Some strains of Bacillus have been shown to be effective in
preventing and controlling disease in poultry (La Ragione et al.,
2001; La Ragione and Woodward, 2003) This is likely in part due to
antimicrobial compounds commonly produced and secreted by many
Bacillus species such as polyketides, lipopeptides and bacteriocins
(Tagg et al., 1976). Five Bacillus strains have been isolated and
selected for their antimicrobial properties against Clostridium
perfringens and APEC. By surveying the level of APEC in the
gastrointestinal tracts of turkeys from a US commercial complex
before and after treatment with the Bacillus DFM product, according
to one embodiment of the present invention, a significant reduction
in APEC after treatment was detected and a maintenance of said
reduction for successive flocks.
[0157] Design. Three gastrointestinal tracts (GIT) from turkeys
were sampled from a variety of houses representing an array of ages
within a commercial turkey complex in three separate sampling
events: one before implementation of the Bacillus DFM product,
according to one embodiment of the present invention, another 10
months after implementation, and final sampling one year and 6
months after implementation. Sampling 1 consisted of 60 GITs,
sampling 2 had 30 GITs, and sampling 3 had 30 GITs.
[0158] Materials and Methods. Direct fed microbial: Treated birds
were given feed supplemented with a formulation of Bacillus strains
1104 (50%) and 1781 (50%) at a final concentration of
1.5.times.10.sup.5 CFU/g of finished feed over the entire
period.
[0159] Processing of Gastrointestinal Tracts: Selected turkeys were
sacrificed and the gastrointestinal tracts from the duodenal loop
to the cloaca were removed and transported in sterile
Whirl-pak.RTM. bags on ice. Upon arrival, 10 cm sections of the
duodenum, jejunum, and ilium were rinsed with .about.5 mL sterile
0.1% peptone broth, cut longitudinally, and combined in a sterile,
filtered whirl-pak bag. 99 mL of sterile 0.1% peptone was added to
the bag then the sections were masticated at 300 rmp, for 1 min.
Serial dilutions were made and pour plated in duplicate with both
CHROMagar.TM. ECC to enumerate total E. coli.
[0160] APEC Screening: Typical E. coli colonies on CHROMagar.TM.
appear blue. After 12-24 h of incubation at 37.degree. C., all blue
colonies were counted and recorded as presumptive APEC CFU/g
counts. Five isolated blue colonies from each bird were picked and
enriched in TSB (Becton, Dickenson & Company, Franklin Lakes,
N.J.) if possible. Genomic DNA was extracted from each isolate
using the following gDNA extraction method: Add 20 .mu.L of
lysozyme (100 mg/mL) to 500 .mu.L of overnight growth in TSB and
incubate at 37.degree. C. for 30 min, add 300 .mu.L of lysis buffer
(6 M Guanidine, 20% Triton-X 100, 10 mM Tris-HCl, pH 7.5) and
incubate at 25.degree. C. for 15 min, add 20 .mu.l of Protease K
800 U/ml (NEB, Ipswich, Mass.) and incubate at 55.degree. for 30
min, transfer 400 .mu.L of lysate to a Wizard.RTM. SV 96 Binding
Plate (Promega, Fitchburg, Wis.)and continue with manufacturer's
filtration instructions from Wizard.RTM. SV 96 Genomic DNA
Purification System starting from step 3.C.4 (4/15/revision)
(Promega, Fitchburg, Wis.).
[0161] APEC pathotype was determined using multiplex polymerase
chain reaction (mPCR). In order to be considered APEC, an E. coli
isolate had to contain at least two of the five following
APEC-associated virulence genes in its genome: hlyF, ompT, iroN,
iss, iutA. Each reaction mixture contained 4 mM magnesium chloride
(Invitrogen, Carlsbad, Calif.), 0.25 mM deoxynucleoside
triphosphates (Invitrogen, Carlsbad, Calif.), 0.25 .mu.M each
primer (Eurofins, Brussels, Belgium), and 1 U Paltinum.RTM. Taq DNA
Polymerase (Invitrogen, Carlsbad, Calif.) and 5 .mu.L of template
gDNA (Johnson et al., 2008). The reaction was run on an Applied
Biosystems Veriti.RTM. Thermal Cycler (ThermoFisher Scientific,
Milwaukee, Wis.) with the following protocol: 94.degree. C. for 2
min; 25 cycles of 94.degree. C. for 30 s, 63.degree. C. for 30 s,
68.degree. C. for 3 min; and a final cycle of 72.degree. C. for 10
min. The mPCR product was then run through capillary gel
electrophoresis using a Fragment Analyzer.TM. from Advance
Analytical Technologies, Inc. and visualized on PROsize 2.0
(Advanced Analytical Technologies, Inc., Ames, Iowa).
[0162] Counts and Statistics: Pathotype levels for each bird were
determined by multiplying the weight-adjusted presumptive CFU/g
counts by the percent of presumptive isolates from each bird that
were revealed to be APEC. Birds that did not produce any detectable
E. coli colonies on agar plates or birds that did not produce any
confirmed APEC strains through screening were assigned a value of 0
CFU/g.
[0163] Statistical significance for the comparison of the three
samplings was determined using a one-way ANOVA multiple analysis.
Significant difference threshold was set at P<0.05.
[0164] Results. The APEC counts represented in CFU/g of tissue are
shown in FIG. 5. Birds tested pre-DFM had an average APEC level of
2.7.times.10.sup.7 CFU/g, while DFM-treated birds from samplings 2
and 3 had significantly lower levels with 1.4.times.10.sup.4 CFU/g
and 7.5.times.10.sup.4 CFU.g, respectively. While the 3.sup.rd
sampling exhibited a higher average level of APEC, this difference
was not significant.
[0165] Discussion. These data demonstrate significant reduction of
APEC levels in turkey GITs fed the Bacillus DFM product, according
to one embodiment of the present invention. They also show that the
APEC inhibitory capacity of the DFM persists over time. Our
research shows that including the Bacillus DFM product, according
to one embodiment of the present invention, in feed is effective in
reducing APEC prevalence in turkeys, therefore decreasing the
disease-burden in commercial turkey operations.
Example 9
Pathogen Levels Rebound After the Bacillus DFM Product, According
to One Embodiment of the Present Invention, is Removed from
Feed
[0166] Introduction. The gastrointestinal-associated pathogens
Clostridium perfringens and avian pathogenic Escherichia coli
(APEC) can have significant negative ramifications on the
productivity of commercial broiler operations (Georgopoulou et al.,
2005) C. perfringens strains that produce alpha toxin are
categorized as the C. perfringens Type A toxinotype and cause
necrotic enteritis in poultry which increases mortality and reduces
weight gain (Immerseel et al., 2004). APEC is a causative agent for
colibacillosis in birds in the form of airsacculitis, cellulitis,
pericarditis, or perihepatitis (Barnes H J et al., 2008).
Colibacillosis infections are of considerable concern for the
poultry industry as they are the responsible for high rates of bird
death and are the most reported reason for processing rejection
(Yogaratnam, 1995). Controlling or reducing rates of NE and
colibacillosis in the commercial broiler industry can increase
efficiency and productivity which may bare substantial economic
impacts for poultry growers. Some strains of Bacillus have been
shown to be effective in preventing and controlling disease in
poultry (La Ragione and Woodward, 2003; La Ragione et al., 2001).
This is likely in part due to antimicrobial compounds commonly
produced and secreted by many Bacillus species (Tagg et al., 1976).
Five Bacillus strains selected for their antimicrobial properties
against Clostridium perfringens and avian pathogenic E. coli (APEC)
were isolated by the inventors. These five Bacillus strains were
commercialized for use as DFM in poultry feed. By surveying
pathogens in the gastrointestinal tracts of broilers from a
commercial complex before, during, and after treatment with the
Bacillus DFM product, according to one embodiment of the present
invention, a significant reduction in both C. perfringens and APEC
during treatment and a numerical increase in APEC was detected, and
a significant increase in C. perfringens after the product was
removed from feed.
[0167] Design. Three gastrointestinal tracts (GIT) from broilers
were sampled from a variety of houses representing an array of ages
within a commercial broiler complex in four separate sampling
events: samplings 1 and 2--before implementation of the Bacillus
DFM product, according to one embodiment of the present invention,
sampling 3--during implementation, and sampling 4--off the Bacillus
DFM product, according to one embodiment of the present invention.
Sampling 1 consisted of 28 GITs, sampling 2 had 30 GITs, sampling 3
had 28 GITs, and sampling 4 had 36 GITs.
[0168] Materials and Methods. Direct fed microbial: Treated birds
were given feed supplemented with a formulation of Bacillus strains
1104 (50%) and 1781 (50%) at a final concentration of
1.5.times.10.sup.5 CFU/g of finished feed.
[0169] Processing of Gastrointestinal Tracts: For each sampling,
selected broilers were sacrificed and the gastrointestinal tracts
from the duodenal loop to the cloaca were removed and transported
in sterile Whirl-pak.RTM. bags on ice. Upon arrival, 10 cm sections
of the duodenum, jejunum, and ilium were rinsed with .about.5 mL
sterile 0.1% peptone broth, cut longitudinally, and combined in a
sterile, filtered whirl-pak bag. 99 mL of sterile 0.1% peptone was
added to the bag then the sections were masticated at 300 rmp, for
1 min. Serial dilutions were made and pour plated in duplicate with
both CHROMagar.TM. ECC to enumerate E. coli, and perfringens TSC
agar base (Oxoid.TM.) with D-cycloserine (Sigma, 400 mg/L) for
Clostridium enumeration.
[0170] APEC Screening: Typical E. coli colonies on CHROMagar.TM.
appear blue. After 12-24 h of incubation at 37.degree. C., all blue
colonies were counted and recorded as presumptive APEC CFU/g
counts. Five isolated blue colonies from each bird were picked and
enriched in TSB (Becton, Dickenson & Company, Franklin Lakes,
N.J.) if possible. Genomic DNA was extracted from each isolate
using the following gDNA extraction method: Add 20 .mu.L of
lysozyme (100 mg/mL) to 500 .mu.L of overnight growth in TSB and
incubate at 37.degree. C. for 30 min, add 300 .mu.L of lysis buffer
(6 M Guanidine, 20% Triton-X 100, 10 mM Tris-HCl, pH 7.5) and
incubate at 25.degree. C. for 15 min, add 20 .mu.l of Protease K
800 U/ml (NEB, Ipswich, Mass.) and incubate at 55.degree. for 30
min, transfer 400 .mu.L of lysate to a Wizard.RTM. SV 96 Binding
Plate (Promega, Fitchburg, Wis.) and continue with manufacturer's
filtration instructions from Wizard.RTM. SV 96 Genomic DNA
Purification System starting from step 3.C.4 (4/15/revision)
(Promega, Fitchburg, Wis.).
[0171] APEC pathotype was determined using multiplex polymerase
chain reaction (mPCR). In order to be considered APEC, an E. coli
isolate had to contain at least two of the five following
APEC-associated virulence genes in its genome: hlyF, ompT, iroN,
iss, iutA. Each reaction mixture contained 4 mM magnesium chloride
(Invitrogen, Carlsbad, Calif.), 0.25 mM deoxynucleoside
triphosphates (Invitrogen, Carlsbad, Calif.), 0.25 .mu.M each
primer (Eurofins, Brussels, Belgium), and 1 U Paltinum.RTM. Taq DNA
Polymerase (Invitrogen, Carlsbad, Calif.) and 5 .mu.L of template
gDNA (Johnson et al., 2008). The reaction was run on an Applied
Biosystems Veriti.RTM. Thermal Cycler (ThermoFisher Scientific,
Milwaukee, Wis.) with the following protocol: 94.degree. C. for 2
min; 25 cycles of 94.degree. C. for 30 s, 63.degree. C. for 30 s,
68.degree. C. for 3 min; and a final cycle of 72.degree. C. for 10
min. The mPCR product was then run through capillary gel
electrophoresis using a Fragment Analyzer.TM. from Advance
Analytical Technologies, Inc. and visualized on PROsize 2.0
(Advanced Analytical Technologies, Inc., Ames, Iowa).
[0172] C. perfringens Type A Screening: Presumptive C. perfringens
isolates appear black on perfringens TSC agar base. All black
colonies were counted and recorded as presumptive C. perfringens
CFU/g counts. Five isolated black colonies from each bird were
picked and grown in RCM broth (Oxoid.TM.) if possible. Genomic DNA
was extracted from each isolate using the Roche Applied Science
High Pure PCR Template Kit.
[0173] C. perfringens toxinotype was determined using polymerase
chain reaction (PCR) to amplify the alpha toxin gene. In order for
an isolate to be considered Clostridium perfringens Type A it had
to contain the alpha toxin gene, otherwise it was categorized as a
non-perfringens Clostridium. Each reaction mixture contained 2.5
.mu.L 10.times.PCR buffer (Invitrogen, Carlsbad, Calif.), 1.6 .mu.L
magnesium chloride (Invitrogen, Carlsbad, Calif.), 0.5 .mu.L
deoxynucleoside triphosphates (Invitrogen, Carlsbad, Calif.), 100
pmol primers (Eurofins, Brussels, Belgium), and 1 U Paltinum.RTM.
Taq DNA Polymerase (Invitrogen, Carlsbad, Calif.) and 2 .mu.L of
template gDNA, 7.8 .mu.L of ddH.sub.20 (Yoo et al., 1997). The
reaction was run on an Applied Biosystems Veriti.RTM. Thermal
Cycler (ThermoFisher Scientific, Milwaukee, Wis.) with the
following protocol: 5 min at 94.degree. C., followed by 30
incubation cycles consisting of 1 min at 55.degree. C., 1 min at
72.degree. C., and 1 min at 94.degree. C. The PCR product was then
run through capillary gel electrophoresis using a Fragment
Analyzer.TM. from Advance Analytical Technologies, Inc. and
visualized on PROsize 2.0 (Advanced Analytical Technologies, Inc.,
Ames, Iowa).
[0174] Counts and Statistics: Pathotype levels for each bird were
determined when the weight-adjusted presumptive CFU/g counts were
multiplied by the ratio of total presumptive to pathogenic isolates
(C. perfringens or APEC) and then log-transformed. Birds that did
not yield any detectable colonies on agar plates or birds that did
not produce any confirmed pathogens through screening were assigned
a value of 0 CFU/g. D
[0175] Statistical significance for the comparison of the four
samplings was determined using a one-way ANOVA multiple analysis.
Significant difference threshold was set at P<0.05.
[0176] Results. The pathogen counts represented in CFU/g of tissue
are shown in FIGS. 6 and 7. APEC levels significantly increased
from sampling 1 to sampling 2 with average APEC levels of
7.0.times.10.sup.2 and 3.0.times.10.sup.4 CFU/g, respectively. APEC
levels were then significantly lowered in the birds treated with a
DFM product, according or one embodiment of the present invention,
in sampling 3 with an average of 1.5.times.10.sup.3 CFU/g and then
increased, though not significantly, in sampling 4 with an average
level of 3.9.times.10.sup.3 CFU/g.
[0177] Clostridium perfringens levels of treated birds in sampling
3 were below detectable limits for all birds, making levels of
treated birds significantly lower than those of sampling 2 and
sampling 4, which exhibited averages of 6.5 and 4.8 CFU/g,
respectively, and numerically lower than sampling 1 which yielded
an average of 1.2 CFU/g.
[0178] Discussion. Comparison of APEC and C. perfringens levels
between sampling 1 and sampling 2 shows a significant upward trend
indicating that flock gastrointestinal health was becoming
increasingly burdened during that time. DFM-treated birds in
sampling 3 showed a significantly decreased enteric pathogen load
(both APEC and C. perfringens), reversing the trend and providing
strong evidence that the Bacillus DFM product, according to one
embodiment of the present invention, has an effective capacity to
inhibit APEC and C. perfringens. Additionally, after the product
was removed from feed (sampling 4), both APEC and C. perfringens
levels increased, supplying further evidence that the Bacillus DFM
product, according to one embodiment of the present invention,
positively modulates the gastrointestinal health of broilers, with
respect to APEC and C. perfringens. Reduction of these pathogens
can reduce cases of disease in broilers such as avian
colibacillosis and necrotic enteritis, diseases which present
significant financial liability to the poultry industry. Our
research shows that including the Bacillus DFM product, according
to one embodiment of the present invention, in feed is effective in
reducing APEC and C. perfringens prevalence in broilers, therefore
decreasing the disease-burden in commercial broiler operations.
Example 10
Intestinal Immunity
[0179] Introduction. Bacillus sp. have been tested as DFMs in
commercial poultry applications and have been shown to improve
performance, positively modulate intestinal microbiota, inhibit
pathogen colonization and improve nutrient digestibility (La
Ragione and Woodward, 2003; Lee et al., 2010; Li et al., 2016;
Nguyen et al., 2015; Park and Kim, 2014, 2015; Sen et al., 2012).
Limited research exists on the effects of Bacillus strains on
alterations in gut immune parameters and the regulation of
intestinal tight junction (TJ) protein expression. The present
study was conducted with the objective of evaluating the effects of
dietary supplementation the Bacillus strains identified herein,
according to the present invention, on performance, gut immune
response and epithelial barrier integrity in broilers.
[0180] Methods. Birds and husbandry: One hundred and forty day-old
male broiler chicks (Ross/Ross) were obtained from a local hatchery
(Longenecker's Hatchery, Elizabethtown, Pa.) and were randomly
allocated to Petersime brooder cages. Cages were equipped with a
separate feeder, water trough and a digitally controlled electrical
heat source. The experimental diets in mash form and tap water were
provided to the chicks ad libitum. Care and management of the birds
followed recommended guidelines (FASS, 2010). All experimental
protocols and procedures were approved by the Small Animal Care
Committee of the Beltsville Agricultural Research Center.
[0181] Experimental design and diets: Brooder cages with chickens
(0 days of age) were randomly assigned to one of the five dietary
treatment groups (4 cages/treatment, total of 28 birds/treatment).
Based on the treatments assigned, chickens were fed either
antibiotic-free basal diets (treatment 1; controls/CON) or basal
diets mixed with either antibiotics or various DFM (treatment 2-5).
The chickens in treatment 2 were given diets supplemented with
bacitracin methylene disalicylate (BMD) at a 50 g/ton inclusion
rate. The birds in the remaining three groups were fed basal diets
supplemented with either Bacillus strain 1781 (treatment 3; PB1), a
combination of strains 1104+747 (treatment 4; PB2) or strains
1781+747 (treatment 5; PB3). For all DFM treatments, the dose
included a total of 1.5.times.10.sup.5 CFU Bacillus/g of feed. For
treatments with 2-strain combinations, each strain composed 50% of
the total CFU count (each strain represents 7.5.times.10.sup.4 CFU
Bacillus/g of feed).
[0182] Body weight and feed intake measurement: The body weight of
each bird was measured and recorded at 7 and 14 days of age. The
feed provided was weighed and recorded throughout the experimental
period. The feed intake and feed conversion ratios (FCR) for each
treatment were calculated. Body weight and FCR data were used as
criteria to assess the performance differences between the
treatments.
[0183] Collection of intestinal samples: Six 14-day-old chickens
were randomly selected from each group and used for the collection
of intestine samples. Birds were euthanized by cervical dislocation
and the intestines were removed immediately. A small section of the
ileum from each bird was collected aseptically and stored in
RNAlater.RTM. (Applied Biosystems, Foster City, Calif.) at
-20.degree. C. for further use.
[0184] Isolation of RNA and reverse transcription: Total RNA was
isolated from the ileum samples stored in RNAlater.RTM. using
TRIzol (Invitrogen, Carlsbad, Calif.) following the manufacturer's
recommendations. Approximately 50 mg of ileal tissue was
homogenized in 1 mL of TRIzol using a hand-held homogenizer
(TissueRuptor; Qiagen Inc., Valencia, Calif.). Chloroform was added
to the homogenized sample. The sample was centrifuged at
12,000.times.g for 15 minutes at 4.degree. C. to allow phase
separation. RNA present in the colorless upper aqueous phase was
then precipitated with 100% isopropanol (Sigma-Aldrich Corp., St.
Louis, Mo.). The RNA pellet was then washed with 75% ethanol
(Sigma-Aldrich Corp. St. Louis, Mo.), air-dried and re-suspended in
RNase-free water. The quantity of RNA was assessed using a NanoDrop
(ND-1000) spectrophotometer (NanoDrop products, Wilmington, Del.)
by measuring the absorbance at 260 nm. RNA purity was evaluated by
measuring the OD260/OD280 ratio (OD=optical density). The eluted
RNA was stored at -80.degree. C. until further use. Total RNA (1
.mu.g) was then reverse transcribed to cDNA using the
QuantiTect.RTM. reverse transcription kit (Qiagen Inc., Valencia,
Calif.). Briefly, the RNA sample was incubated with gDNA wipeout
buffer at 42.degree. C. for 2 minutes to remove any genomic DNA
contamination. Reverse transcription (RT) of the gDNA-depleted
sample was then carried out by the addition of Quantiscript Reverse
Transcriptase, Quantiscript RT buffer, and RT primer mix (Qiagen
Inc., Valencia, Calif.). The reaction was carried out in a thermal
cycler (Mastercycler.RTM. EP Gradient S; Eppendorf, Hauppauge,
N.Y.); cycling conditions were 42.degree. C. for 30 min, followed
by the inactivation of reverse transcriptase at 95.degree. C. for 3
min. The cDNA samples were divided into aliquots and stored at
-20.degree. C.
[0185] Gene expression analysis by quantitative real-time PCR
(qRT-PCR): The oligonucleotide primer sequences used for qRT-PCR
are shown below in Table 10.
TABLE-US-00010 TABLE 10 Oligonucleotide primer sequences for
qRT-PCR PCR pro- duct Target size Type gene Primer sequence*
(5'-3') (Kb) Reference GAPDH F-GGTGGTGCTAAGCGTGTTAT 264
R-ACCTCTGCCATCTCTCCACA Proin- IL1.beta. F-TGGGCATCAAGGGCTACA 244
flam- R-TCGGGTTGGTTGGTGATG matory IL6 F-CAAGGTGACGGAGGAGGAC 254
R-TGGCGAGGAGGGATTTCT IL8 F-GGCTTGCTAGGGGAAATGA 200
R-AGCTGACTCTGACTAGGAAACTGT IL17F F-TGAAGACTGCCTGAACCA 117
R-AGAGACCGATTCCTGATGT TNFSF15 F-CCTGAGTATTCCAGCAACGCA 292
R-ATCCACCAGCTTGATGTCACTAAC Th1 IL2 F-TCTGGGACCACTGTATGCTCT 256
R-ACACCAGTGGGAAACAGTATCA IFN.gamma. F-AGCTGACGGTGGACCTATTATT 259
R-GGCTTTGCGCTGGATTC Th2 IL4 F-ACCCAGGGCATCCAGAAG 258
R-CAGTGCCGGCAAGAAGTT IL13 F-CCAGGGCATCCAGAAGC 256
R-CAGTGCCGGCAAGAAGTT Regula- IL10 F-CGGGAGCTGAGGGTGAA 272 tory
R-GTGAAGAAGCGGTGACAGC TJ Occludin F-GAGCCCAGACTACCAAAGCAA 68
proteins R-GCTTGATGTGGAAGAGCTTGTTG ZO1 F-CCGCAGTCGTTCACGATCT 63
R-GGAGAATGTCTGGAATGGTCTGA JAM2 F-AGCCTCAAATGGGATTGGATT 59
R-CATCAACTTGCATTCGCTTCA Mucin MUC2 F-GCCTGCCCAGGAAATCAAG 59
R-CGACAAGTTTGCTGGCACAT *F = Forward primer; R = Reverse primer
[0186] The various cytokines and intestinal tight junction proteins
whose differential expression was evaluated in the ileum include
interleukin (IL)1.beta., IL2, IL4, IL6, IL8, IL10, IL13, IL17F,
interferon (IFN).gamma., tumor necrosis factor superfamily
(TNFSF)15, junctional adhesion molecule (JAM)2, occludin, zona
occludens (ZO)1, and mucin2 (MUC2). The primer sequences of TJ
proteins and MUC2 were adapted from Chen et al., 2015.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the
reference gene. Amplification and detection were carried out using
the Stratagene Mx3000P qPCR system (Agilent Technologies Inc.,
Santa Clara, Calif.) and the RT.sup.2 SYBR Green qPCR master mix
(Qiagen). Each sample was analyzed in triplicate and nonspecific
primer amplification was assessed by the inclusion of no template
controls. Standard curves were generated using log.sub.10 diluted
RNA and the levels of individual transcripts were normalized to
those of GAPDH using the Q-gene program (Muller et al., 2002).
[0187] Data analysis: Analysis of data was carried out using
one-way ANOVA with SAS software (version 9.4, SAS Institute Inc.,
Cary, N.C.). Results with a P-value.ltoreq.0.05 were considered as
significantly different. Mean separations were carried out using
Duncan's multiple range test. All data were expressed as the
mean.+-.SEM for each treatment.
[0188] Results. The body weight and FCR results at 14 days of age
are presented in FIGS. 8 and 9, respectively. The birds fed diets
with antibiotic (BMD) and one of the DFM strains, according to the
present invention, (PB1) showed significantly higher body weights
compared to those fed a basal diet (CON). There were no body weight
differences in chickens fed diets supplemented with Bacillus strain
combinations (PB2, PB3). The FCR was found to be significantly
reduced in all chickens that were administered DFM or antibiotic
treatments compared to the controls.
[0189] The mean normalized expression of various pro-inflammatory
cytokines in the ileum are shown in FIG. 10. No differences were
observed in the expression of IL1.beta. and IL17F in any of the
treatment groups receiving supplemented diets compared to controls.
The levels of IL6 were found to be elevated in birds administered
BMD, PB1 and PB2 treatments. IL8 expression was significantly
increased in the PB2 group compared to controls. The birds fed with
DFM (PB1, PB2, PB3) showed significantly increased TNFSF15
expression in the ileum compared to those given non-supplemented
basal diets (CON).
[0190] The expression levels of various Th1 and Th2 cytokines in
the ileum are presented in FIG. 11. IL2 and IL10 were found to be
significantly elevated in PB2 and PB3 treatments compared to
controls. The expression of IL4 was increased only in Bacillus
strain 1781 supplemented birds (PB1). IL13 was increased in birds
given antibiotic (BMD) or DFM (PB1, PB2, PB3) supplemented diets
compared to controls. No changes were observed in the expression of
IFN.gamma. among the various treatment groups.
[0191] The expression of intestinal tight junction protein
genes-JAM2 and ZO1 was significantly increased in the PB2 and PB3
groups, whereas occludin was found to be elevated in the PB2 and
PB3 groups compared to the CON group. Neither the DFM nor
antibiotic supplementation altered MUC2 expression in the ileum at
14 days of age as shown in FIG. 12.
[0192] Discussion. The results show that DFM (PB1)-supplemented
chickens have significantly higher body weights at 14 days of age
compared to non-supplemented controls and the increase in body
weight observed was similar to that of antibiotic- antibiotic-fed
chickens (BMD). The FCR was found to be significantly improved in
all the supplemented groups (BMD, PB1, PB2 and PB3) compared to
controls.
[0193] IL8 (CXCLi2), a chemokine and an important mediator of
innate immune defense, was found to be elevated in PB2 birds.
TNFSF15, a cytokine involved in the differentiation and
proliferation of immune cells was found to be elevated in all
DFM-fed groups (PB1, PB2, PB3. Dietary supplementation with either
Bacillus strain 1781 (PB1), a combination of Bacillus strain
1104+747 (PB2) or antibiotic (BMD) significantly increased the
ileal IL6 expression in broiler chickens.
[0194] In addition to the changes in the expression of various
pro-inflammatory cytokines, this study also investigated the
alterations occurring in T-helper (Th)1 (IL2, IFN.gamma.), Th2
(IL4, IL13) and regulatory cytokines (IL10) in the gut following
Bacillus-DFM supplementation. No differences were observed in IL2
and IFN.gamma. expression. IL4 was found to be upregulated in the
PB1 group compared to controls. IL13 expression was significantly
increased in all DFM (PB1, PB2, and PB3) and antibiotic (BMD)-fed
broilers compared to those fed basal diets (CON). In this study,
IL10 was found to be upregulated in chickens fed diets with
mixtures of DFM strains (PB2, PB3).
[0195] The effects of Bacillus supplementation on the expression of
various intestinal TJ proteins was also investigated. The
expression of occludin was found to be elevated in PB1 and PB2
groups and ZO1 and JAM2 were found to be elevated in the PB2 and
PB3 groups compared to controls (CON). Increased TJ protein
expression in chickens fed DFM-supplemented diets translates to
increased intestinal barrier function and optimal gut health.
[0196] Conclusions. This study documented the immunomodulatory
activities of Bacillus strains in the ileum coupled with changes in
the intestinal TJ proteins. From these results, it can be concluded
that supplementation of broiler diets with Bacillus DFM influences
a diverse array of immune gut barrier functions.
[0197] It should be understood that the above description, while
indicating representative embodiments of the present invention, is
given by way of illustration and not of limitation. Many changes
and modifications may be made within the scope of the present
invention without departing from the spirit thereof, and the
invention includes all such modifications.
[0198] Various additions, modifications and rearrangements are
contemplated as being within the scope of the following claims,
which particularly point out and distinctly claim the subject
matter regarded as the invention, and it is intended that the
following claims cover all such additions, modifications and
rearrangements.
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Sequence CWU 1
1
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Synthetic primer 1gtagacccgt 10220DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 2agagtttgat ymtggctcag
20316DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 3taccttgtta ygactt 16410DNAArtificial
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4gtttcgctcc 10520DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 5accaacatca ttgcggctac 20624DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
6acctttgtag aagcagcaat ttca 24720DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 7tcatacggaa tggcctgggg
20821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 8acttttgttg aagttggccc g 21921DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
9acagttgctg ttagtgtccc a 211020DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 10gacgatatcg gttcctgcgt
201120DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 11cacatcaatc tggggcaagc 201224DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
12tcatatcaac taagtgtagc cgca 241320DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
13agacgttacg ttttccccct 201420DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 14cgggccaact tcaacaaaag
201520DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 15ggtggtgcta agcgtgttat 201620DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
16acctctgcca tctctccaca 201718DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 17tgggcatcaa gggctaca
181818DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 18tcgggttggt tggtgatg 181919DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
19caaggtgacg gaggaggac 192018DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 20tggcgaggag ggatttct
182119DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 21ggcttgctag gggaaatga 192224DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
22agctgactct gactaggaaa ctgt 242318DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
23tgaagactgc ctgaacca 182419DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 24agagaccgat tcctgatgt
192521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 25cctgagtatt ccagcaacgc a 212624DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
26atccaccagc ttgatgtcac taac 242721DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
27tctgggacca ctgtatgctc t 212822DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 28acaccagtgg gaaacagtat ca
222922DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 29agctgacggt ggacctatta tt 223017DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
30ggctttgcgc tggattc 173118DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 31acccagggca tccagaag
183218DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 32cagtgccggc aagaagtt 183317DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
33ccagggcatc cagaagc 173418DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 34cagtgccggc aagaagtt
183517DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 35cgggagctga gggtgaa 173619DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
36gtgaagaagc ggtgacagc 193721DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 37gagcccagac taccaaagca a
213823DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 38gcttgatgtg gaagagcttg ttg 233919DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
39ccgcagtcgt tcacgatct 194023DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 40ggagaatgtc tggaatggtc tga
234121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 41agcctcaaat gggattggat t 214221DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
42catcaacttg cattcgcttc a 214319DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 43gcctgcccag gaaatcaag
194420DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 44cgacaagttt gctggcacat 20
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References